CN118581146A - A protein that effectively increases the yield of Arabidopsis/rice under water-saving or drought conditions and the application of its encoding gene - Google Patents

Abstract

The present invention provides a protein that effectively improves the yield of Arabidopsis/rice under water-saving or drought conditions and the application of its encoding gene, which belongs to the field of biotechnology, and specifically discloses the application of Arabidopsis/rice UBP15 protein and its encoding gene to avoid growth inhibition, reduced tillering, reduced seeds and reduced yield under drought conditions. The present invention studies the function of the UBP15 gene in Arabidopsis (dicotyledonous leaves) and rice (monocotyledonous leaves), and finds that gene editing mutation ubp15 can effectively improve the yield, traits and phenotype of Arabidopsis/rice under drought conditions, and significantly increase yield, which helps to understand the molecular mechanism of Arabidopsis/rice responding to drought to regulate yield and traits, provides an effective target gene for precise improvement of crop gene editing, and further provides a potential feasible strategy for the creation of water-saving crop germplasm.

Description

A protein that effectively increases the yield of Arabidopsis/rice under water-saving or drought conditions and the application of its encoding gene

Technical Field

The invention belongs to the field of biotechnology, relates to plant transgenic biotechnology breeding, and in particular to a protein and an application of a coding gene thereof for effectively improving the yield of Arabidopsis/rice under water-saving or drought conditions.

Background Art

As the global climate warms, adversities such as drought are becoming more severe year by year. According to data reported in 2023 by the 101 contracting parties to the United Nations Convention to Combat Desertification (UNCCD), 1.84 billion people are currently threatened by drought, and the area and severity of drought are positively correlated with global warming. In 2022, the area affected by drought in Europe was almost four times the area affected in 2000, reaching 630,000 square kilometers. In my country, arid/semi-arid areas account for 42% of the country's land area, and as the global climate warms, the frequency and severity of droughts are also increasing year by year.

Drought leads to a decrease in crop yields, which seriously threatens food security. However, there is still a lack of research on key genes that can avoid crop yield reduction under drought conditions. Drought-induced seed filling in plants is inhibited, resulting in small seeds. At the same time, the number of tillers and seeds of plants under drought conditions also decreases significantly, resulting in a significant decrease in plant yield under drought conditions, or even no yield. Although this yield reduction is related to the weakening of photosynthesis under drought conditions, the intrinsic adaptive regulatory mechanism of plants also plays a key role in the reduction of yield under adverse environments such as drought. In recent years, the mining of key genes to avoid crop yield reduction under high temperature, saline-alkali and other conditions has received widespread attention from the society. For example, the TT3 and AT1 genes can effectively avoid crop yield reduction under high temperature and saline-alkali conditions, suggesting that the yield under adverse environments is affected by the intrinsic regulatory pathways of plants. Similarly, the phenomenon of small seeds under drought conditions is also considered to be an escape adaptation response of plants to drought. Since small seeds are conducive to shortening the seed development cycle, they can use seeds, which have extremely strong stress resistance, to escape from adverse environments such as drought. However, the molecular mechanism of plant response to drought to regulate yield traits is still unclear, and there is still a lack of effective genes to avoid crop yield reduction under drought conditions.

Molecular design precision breeding is a new breeding direction in recent years, especially gene editing breeding, which has become a more widely accepted precision breeding method in the market. Compared with transgenic breeding, gene editing breeding can modify genes without residual transgenic components, so it has become a new hot spot in molecular breeding in recent years. Gene editing breeding generally requires the clarification of the important regulatory functions of the targeted gene, and the gene can change the gene function through gene editing. Therefore, the research on functional genes that can be used for gene editing and modification for high and stable yields under drought is still relatively scarce.

Summary of the invention

In response to the above problems, the present invention provides a protein and its encoding gene that effectively improves the yield of Arabidopsis/rice under water-saving or drought conditions, providing a target gene and a feasible solution for improving the water-saving ability of crops by gene editing and other methods.

To achieve the above purpose, the technical solution adopted by the present invention is:

A protein that effectively increases the yield of Arabidopsis/rice under water-saving or drought conditions, wherein the protein is a conserved homologous protein UBP15 of Arabidopsis/rice that is used to prevent growth inhibition, seed shrinkage, reduced tillering, and reduced yield of Arabidopsis/rice under drought conditions;

Arabidopsis/rice conserved homologous proteins UBP15 include Arabidopsis UBP15 (AT1G17110) and rice UBP15 (Os02g0244300/LOC_Os02g14730);

The amino acid sequence of Arabidopsis thaliana UBP15 is shown in SEQ ID NO: 1;

The amino acid sequence of rice UBP15 is shown in SEQ ID NO: 2;

The conserved homologous protein UBP15 of angiosperms includes both USP domain (Prosite number: PS00972/PS50235 or ProRule number: PRU01035) and CDU15 domain, including but not limited to Arabidopsis thaliana UBP15 (At1g17110), rice UBP15 (LOC_Os02g14730), homologous proteins of other species or proteins encoded by other artificial genes;

Genes encoding both the characteristic amino acid domains USP and CDU15, including but not limited to the Arabidopsis thaliana UBP15 (At1g17110) gene, the rice UBP15 (LOC_Os02g14730) gene, homologous genes of other species or other artificial genes;

The gene sequence of Arabidopsis thaliana UBP15 is shown in SEQ ID NO: 3;

The gene sequence of rice UBP15 is shown in SEQ ID NO: 4;

The CDS sequence of the Arabidopsis thaliana UBP15 gene is shown in SEQ ID NO: 5;

The CDS sequence of the rice UBP15 gene is shown in SEQ ID NO: 6;

>USP domains are:

GL(I)V(X)NCGNSCYANAV(A)LQS(O)LT(M)C(X)TKPLV(X)A(I/V)Y(F/H)LLR(X)RS(L)HSR( K)S(X)C(S/Y)S(C/Y)G(X)K(R)D(N)WCLM(V)CELEQ(K/R)H(Y)V(A)M( S)M(T)LR(K)ES(X)GG(X)PL(V)SA(P)S(N)R(K)I(F)LS(L)H(Q/R)M( I /L)R(Q)S(N/G)IN(G)C(S/G)Q(RH)I(L/M)GD(X)GSQEDAHEFLRL(H)L(I)V(I)A (M)SMQS(G/A)I(X)CLE(D)R(G/A)L(Q)GGET(X)KV(I)D(E/N)P(X)R(S/I )L(Q)QE(D/Q)TTL(F)V(I)QH(Q)M(T/I)FGGR(Q)LR(KQ)SKVKCL(Q)R(N)CD(X) H(L/V)ESE(A)R(C)Y(H/S)EN(S)IMDLT(S)LEIY(X)GW(R)VE(Q)SLQ(E)DALTQFTR(X)PED( E)LDGE(D)NMYR(K)CS(G)R(S)CA(X)G(X)YVR(K/E)AR(Q)KE(Q)LS(C)I(V)H( Q)EA(V)PNILTI(V)VLKRFQ(K)E(X)GR(X)YGKINKCI(V)S(T/A) FPE(D)MLDMI(V)P(Y)FM(V)TR(G)T(A/S)G(D/A)DV(X)PPLYM(F)LYAVI(V)VHL(V)DT( E)LNASFSGHYI(V)S(A/T)YV(I)KDL(M)R(Q/H)GN(T)WY(X)RI(V)DDS(T)E(K)I(V) H(Q/K)P(X)VP(X)M(X)T(X)Q(R)VMS(T)EGAYM(I)LFYM(X)RS;

Note: The amino acids in parentheses are possible alternative amino acid residues before the parentheses, and "X" indicates that there are three or more alternative amino acid residues;

>The CDU15 domains are:

SE(D)WS(H)LFTSSDE(D)S(A)SFT(S)TEST(.)RDSFSV(T)V(I/A)DY;

Note: The amino acids in parentheses are possible replacement amino acid residues before the parentheses. "(.)" indicates that the "T" before the parentheses may be missing.

The full name of the CDU15 domain is "Conserved-Domain of UBP15", and its Chinese name is "UBP15 conserved domain";

The CDU15 domain is a characteristic sequence of the UBP15 protein or the product encoded by the UBP15 gene, and can be used to characterize UBP15 homologous proteins in different species.

Furthermore, the drought condition includes any of the following:

The Arabidopsis/rice plant is deprived of water in the late vegetative growth stage or early reproductive growth stage until the Arabidopsis/rice plant is completely dried up;

Or, when Arabidopsis/rice is deprived of water in the late vegetative growth stage or early reproductive growth stage, the wilting of Arabidopsis/rice is a sign;

Alternatively, under water-saving treatment in the late vegetative growth stage or early reproductive growth stage of Arabidopsis/rice, the phenotype of significant moisture reduction, such as cracking of field soil, is marked.

A function of a gene encoding the above protein that effectively increases the yield of Arabidopsis thaliana/rice under water-saving or drought conditions, wherein the gene encoding the above protein that effectively increases the yield of Arabidopsis thaliana/rice under water-saving or drought conditions is the UBP15 gene;

The function is that the UBP15 gene regulates growth, seed size, tiller/branch number and yield in response to drought in plants.

Furthermore, the function is to obtain stable yield by mutating/knocking down the UBP15 gene, thereby avoiding growth inhibition, seed shrinkage, reduction in tillering/branching number and yield reduction of Arabidopsis/rice under drought conditions.

Furthermore, the function is to avoid growth inhibition, seed shrinkage, reduction in tillering/branching number, and yield reduction of Arabidopsis/rice under drought conditions by reducing the UBP15 protein level, inhibiting the UBP15 protein function, or knocking out/knockdown the UBP15 gene, thereby achieving a stable yield;

The Arabidopsis/rice containing the UBP15 gene has no obvious change in seed size under drought conditions relative to good watering conditions, the number of tillers/branches does not decrease significantly, or even increases, the growth inhibition is relatively reduced, and the relative decrease in seed yield is not obvious.

Furthermore, the UBP15 gene knockout is performed by any of the following techniques:

Knockout of the UBP15 gene using T-DNA insertion;

Knock out the UBP15 gene using gene editing, such as CRISPR/Cas, ZFN, TALEN and other gene editing technologies;

Knock out the UBP15 gene by chemical mutagenesis, such as mutagenesis treatment with chemical mutagens such as EMS;

Knocking out the UBP15 gene by physical mutagenesis, such as space radiation, alpha radiation, beta radiation, neutrons and other particles, ultraviolet radiation, and microwave radiation;

The UBP15 gene knockdown was performed using any of the following techniques:

Knock down the UBP15 gene using gene editing, such as CRISPR/Cas, ZFN, TALEN and other gene editing technologies;

Knock down the UBP15 gene using gene silencing technology, including RNAi interference technology and other gene silencing technologies;

Knockdown of UBP15 gene by promoter changes, such as gene editing promoter sequence, T-DNA insertion promoter sequence, etc.

Knockdown of UBP15 gene by modifying key factors that regulate UBP15 gene expression;

Knock down the UBP15 gene using chemical reagents or physical factors;

The reduction of UBP15 protein level or inhibition of UBP15 protein function is achieved by any of the following techniques:

Reduction of UBP15 protein levels by effective molecular treatment;

The UBP15 protein level is reduced by effective environmental treatments such as drought;

Reduction of UBP15 protein levels caused by other protein manipulation techniques;

Inhibition of UBP15 function caused by protein modification, molecular interaction, inhibitors, etc.

Drought conditions include any of the following:

Cut off water in the late vegetative growth stage or early reproductive growth stage of plants until the plants completely dry up and die;

Or, when the plant is deprived of water in the late vegetative growth stage or early reproductive growth stage, the plant will wilt as a sign;

Alternatively, under water-saving treatment in the late vegetative growth stage or early reproductive growth stage of plants, the phenotype may be marked by a significant decrease in moisture content, such as cracking of the field soil.

An application of a gene encoding the above protein that effectively increases the yield of Arabidopsis thaliana/rice under water-saving or drought conditions, wherein the gene encoding the above protein that effectively increases the yield of Arabidopsis thaliana/rice under water-saving or drought conditions is the UBP15 gene;

The application is the application of the UBP15 gene in regulating growth, seed size, tillering/branching number and yield of Arabidopsis/rice in response to drought.

Furthermore, the application is to utilize UBP15 gene knockout, UBP15 gene knockdown, UBP15 protein level reduction or UBP15 protein function inhibition to inhibit the growth of Arabidopsis thaliana/rice under drought conditions, reduce seeds, reduce tillering/branching numbers and reduce yield, thereby promoting high and stable yield of Arabidopsis thaliana/rice under drought conditions;

Through the above-mentioned means, the seed size of Arabidopsis thaliana/rice containing the UBP15 gene under drought conditions will not change significantly relative to control conditions, the number of tillers/branches will not decrease significantly, or may even increase, the growth inhibition will be relatively reduced, and the seed yield will not decrease significantly, ultimately enabling Arabidopsis thaliana/rice to have high and stable yields under drought conditions.

Furthermore, the UBP15 gene knockout is performed by any of the following techniques:

Knockout of the UBP15 gene using T-DNA insertion;

Knock out the UBP15 gene using gene editing, such as CRISPR/Cas, ZFN, TALEN and other gene editing technologies;

Knock out the UBP15 gene by chemical mutagenesis, such as mutagenesis treatment with chemical mutagens such as EMS;

The UBP15 gene is knocked out by physical mutagenesis, such as space rays, alpha rays, beta rays, neutrons and other particles, ultraviolet radiation, and microwave radiation.

Furthermore, the UBP15 gene knockdown is performed by any of the following techniques:

Knock down the UBP15 gene using gene editing, such as CRISPR/Cas, ZFN, TALEN and other gene editing technologies;

Knock down the UBP15 gene using gene silencing technology, including RNAi interference technology and other gene silencing technologies;

Knockdown of UBP15 gene by promoter changes, such as gene editing promoter sequence, T-DNA insertion promoter sequence, etc.

Knockdown of UBP15 gene by modifying key factors that regulate UBP15 gene expression;

Knock down the UBP15 gene using chemical reagents or physical factors.

Furthermore, the reduction of UBP15 protein level or inhibition of UBP15 protein function is achieved by any of the following techniques:

Reduction of UBP15 protein levels by effective molecular treatment;

The UBP15 protein level is reduced by effective environmental treatments such as drought;

Reduction of UBP15 protein levels caused by other protein manipulation techniques;

UBP15 function inhibition caused by protein modification, molecular interaction, inhibitors, etc.

Furthermore, the drought condition includes any of the following:

The Arabidopsis/rice plant is deprived of water in the late vegetative growth stage or early reproductive growth stage until the Arabidopsis/rice plant is completely dried up;

Or, when Arabidopsis/rice is deprived of water in the late vegetative growth stage or early reproductive growth stage, the wilting of Arabidopsis/rice is a sign;

Alternatively, under water-saving treatment in the late vegetative growth stage or early reproductive growth stage of Arabidopsis/rice, the phenotype of significant moisture reduction, such as cracking of field soil, is marked.

Furthermore, the application is to prevent the growth inhibition, seed shrinkage, reduction in tillering/branching number and yield reduction of Arabidopsis/rice under drought conditions by knocking out/knockdown of the UBP15 gene, thereby promoting high and stable yield of Arabidopsis/rice under drought conditions.

Furthermore, the UBP15 gene knockout/knockdown is achieved by using gene editing, T-DNA insertion destruction, chemical/physical radiation mutagenesis to cause UBP15 gene mutation, or by using gene silencing, transcriptional regulation and manipulation technology to inhibit UBP15 gene expression, or by using inhibitors, protein modification and manipulation technology to inhibit UBP15 protein activity.

The beneficial effects of the application of a protein for effectively increasing plant yield under water-saving or drought conditions and its encoding gene of the present invention are:

Through the study of the function of the UBP15 gene in Arabidopsis (dicotyledon) and rice (monocotyledon), the present invention found that the gene editing mutation UBP15 can effectively improve the yield, traits and phenotype of plants under drought conditions, and significantly increase yield, which helps to understand the molecular mechanism of plant response to drought to regulate yield and traits, and provides an effective target gene for the precise improvement of crop gene editing, and further provides a potential feasible strategy for the creation of water-saving crop germplasm;

The present invention provides a protein that effectively increases the yield of plants under water-saving or drought conditions and the application of its encoding gene. By mutating or editing the gene, growth inhibition, reduced tillering, smaller seeds and reduced yield of plants under drought conditions can be effectively avoided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a comparative diagram of branching, seed size and yield of Arabidopsis thaliana ubp15 mutants under drought in Examples 1-2 of the present invention; wherein FIGa is a comparative diagram of Col-0 plants, ubp15-1 plants and ubp15-2 plants under watering control and drought treatment in Example 1; FIGb is a comparative diagram of seed yield of each individual plant of Col-0 plants, ubp15-1 plants and ubp15-2 plants under watering and drought conditions in Example 1; FIGc is a comparative diagram of myc-UBP15 protein accumulation in 35S::myc-UBP15 transgenic plants under watering and drought conditions in Example 2; FIGd is a comparative diagram of seed size of each individual plant of Col-0 plants, ubp15-1 plants and ubp15-2 plants under watering and drought conditions in Example 1; FIGe is a comparative diagram of seed size of each individual plant of Col-0 plants, ubp15-1 plants and ubp15-2 plants in Example 1; Comparison of the seed size statistics of each individual plant between ubp15-1 and ubp15-2 plants under watering and drought conditions;

FIG2 is a comparison of amino acid sequences of some homologous genes containing the amino acid characteristic sequence of UBP15 in Arabidopsis thaliana and common crops in Example 3 of the present invention; wherein Zf-MYND represents the Zf-MYND domain; USP represents the USP domain; CDU15 represents the CDU15 domain;

FIG3 is a rice UBP15 gene knockout mutant constructed in Example 5 of the present invention; wherein, FIGa is a first exon of the UBP15 gene (LOC_Os02g14730) knocked out using the CRISPR-Cas9 technology in the ZH11 rice variety background using the targeting sequence 5'-ATGGGTCTTAAAGAAGGTCCTGG-3'; FIGb is a homozygous mutant of the ubp15 gene editing without transgenic components (i.e., both alleles are missing GACC and ACCAA is inserted);

Figure 4 is a comparison diagram of the regulation of branching, seed size and yield of rice ubp15 gene editing knockout mutation under potted plants and drought conditions in Example 6 of the present invention; wherein, Figure a is a comparison diagram of the homozygous mutant plants of the ubp15 gene editing without transgenic components and the wild-type Zhonghua ZH11 plants under watering and drought conditions in Example 6; Figure b is a comparison diagram of the millet yield of each single plant of the homozygous mutant plants of the ubp15 gene editing without transgenic components and the wild-type Zhonghua ZH11 plants under watering and drought conditions in Example 6; Figure c is a comparison diagram of the millet yield of each single plant of the homozygous mutant plants of the ubp15 gene editing without transgenic components and the wild-type Zhonghua ZH11 plants under watering and drought conditions in Example 6. Figure d is a comparison of the effective tiller numbers of each plant between the edited homozygous mutant plant and the wild-type Zhonghua ZH11 plant under watering and drought conditions; Figure d is a comparison of the seed sizes of each plant between the non-transgenic component ubp15 gene-edited homozygous mutant plant and the wild-type Zhonghua ZH11 plant under watering and drought conditions in Example 6; Figure e is a comparison of the 100-grain weight of each plant between the transgenic component ubp15 gene-edited homozygous mutant plant and the wild-type Zhonghua ZH11 plant under watering and drought conditions in Example 1; in Figures a-e, ZH11 represents the wild-type Zhonghua ZH11 plant, and ubp15 represents the non-transgenic component ubp15 gene-edited homozygous mutant;

Figure 5 is a comparison diagram of the regulation of branching and yield of the rice ubp15 gene-edited knockout mutant under field water-saving conditions in Example 7 of the present invention; wherein, Figure a is a comparison diagram of the ubp15 gene-edited homozygous mutant plant without transgenic components and the wild-type Zhonghua ZH11 plant under irrigation and water-saving conditions in Example 7; Figure b is a comparison diagram of the effective branching of each single plant between the transgenic component ubp15 gene-edited homozygous mutant plant and the wild-type Zhonghua ZH11 plant under irrigation and water-saving conditions in Example 7. Figure a is a comparison chart of the tiller number; Figure c is a comparison chart of the millet yield of each individual plant between the homozygous mutant plant of the ubp15 gene editing without the transgenic component and the wild-type Zhonghua ZH11 plant under irrigation and water-saving conditions in Example 7; Figure d is a statistical comparison chart of the millet yield of each individual plant between the homozygous mutant plant of the ubp15 gene editing without the transgenic component and the wild-type Zhonghua ZH11 plant under irrigation and water-saving conditions in Example 7; In Figures a-d, ZH11 represents the wild-type Zhonghua ZH11 plant, and ubp15 represents the homozygous mutant of the ubp15 gene editing without the transgenic component.

DETAILED DESCRIPTION

The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

In addition, the experimental methods in the specific examples disclosed below are all conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

Arabidopsis thaliana ubp15-1 (SALK_018601) T-DNA knockout mutant seeds and ubp15-2 (SALK_015611) T-DNA knockout mutant in the specific examples disclosed below were ordered from the Nottingham Arabidopsis Germplasm Centre (NASC);

Rice ubp15 gene editing knockout mutant was purchased from Biogen.

The data in the specific examples disclosed below were analyzed in GraphPad Prism 8 software, and the two-way ANOVA was used to analyze the two-factor data using Tukey's test mode.

Example 1: Drought treatment of Arabidopsis ubp15 T-DNA knockout mutant

Order SALK_018601 ( ubp15-1 ) T-DNA knockout mutant seeds (i.e., rice ubp15 gene editing knockout mutant) and SALK_015611 ( ubp15-2 ) T-DNA knockout mutant seeds (i.e., rice ubp15 gene editing knockout mutant) from the Search Stock section of NASC (https://arabidopsis.info/).

After the seeds arrived, the mutant seeds and the wild-type Col-0 were planted in culture soil (the culture soil was made by mixing 2/3 humus and 1/3 vermiculite), and cultured for 3 weeks at 22°C and long daylight (16h light/8h dark, light source is 4 T8 fluorescent lamps). One fully expanded leaf was taken, and the mutant genomic DNA and wild-type Col-0 genomic DNA were extracted using the Plant Genomic DNA Rapid Extraction Kit (Biolabo WH0024), and the homozygous mutant was identified and confirmed using the three-primer method (http://signal.salk.edu/tdnaprimers.2.html):

First, primers were queried and synthesized. The primer pair for ubp15-1 identification was SALK_018601 LP: 5′-TTGTGTCATAATGACCCCCTC-3′ and SALK_018601 RP: 5′-TGAGACTTTTTGGGGAATGTG-3′;

The primer pair for ubp15-2 identification was SALK_015611 LP: 5′-TATCAAGTGAGCGTCCCTGAC-3′ and SALK_015611 RP: 5′-TCACACCTCAGGCATTTAACC-3′;

T-DNA intermediate primer LBb1.3: 5’-ATTTTGCCGATTTCGGAAC-3’.

The following PCR identification system (2×taq mix: 5.0μL; LP: 0.2μL; RP: 0.2μl; LBb1.3: 0.2μL; extracted corresponding genomic DNA: 1.0μL; ddH ₂ O: 3.4μL) and amplification program (95℃: 5min; 95℃: 30s, 55℃: 30s, 72℃: 1min, 30 cycles; 72℃: 5min; 16℃: 10min) were used to amplify the corresponding genomic DNA. The wild-type Col-0 genomic DNA was used as the control template. The products after PCR amplification were subjected to gel electrophoresis experiment (120V, 20min) and detected. If the product is a band of about 900bp, it is wild type; if it is two bands, one of 900bp and one of 400-700bp, it is a heterozygote; if it is a band between 400-700bp, it is a homozygous mutant. After selecting the homozygous mutants of ubp15-1 (SALK_018601) and ubp15-2 (SALK_015611) for breeding, the obtained ubp15-1 seeds and ubp15-2 seeds were used for subsequent experiments.

After the surface of the seeds were disinfected (20% 84 bleach solution + 0.1% Tween 20 by volume was used to disinfect the seeds for 7-10 minutes, and then washed 6 times with sterilized water), they were spread on MS plates. One ubp15-1 mutant seedling and one ubp15-2 mutant seedling that had grown normally for 7 days on MS medium were planted in a pot together with two wild-type Col-0 plants. A total of 60 pots were planted, divided into 4 trays of plants. Watering and fertilization were carried out normally (watering once every 4 days, and a small amount of water-soluble fertilizer with a commercial name of Huawuque was applied every other watering) until the plants began to bloom (about 40 days). Two trays of plants were deprived of water and drought treatment, and the flowers that opened after 4 days of deprivation of water were marked with a thin line, so as to later count the seed-related data in the siliques developed from these flowers, and to avoid other interference factors during the experiment. The other two trays of plants were watered normally as a watering control.

After about 14 days of drought without water, the two trays of Arabidopsis plants in the water-deprivation treatment had died of drought, while the Arabidopsis plants in the control treatment grew normally.

Col-0 plants, ubp15-1 plants and ubp15-2 plants under watering control and drought treatment were photographed. The results are shown in Figure 1a. The growth of ubp15-1 plants and ubp15-2 plants under water-free drought treatment was significantly better than that of Col-0 plants under drought treatment, and the number of branches and siliques was significantly greater, while ubp15-1 plants and Col-0 plants grew normally under watering control conditions.

After the seeds of the watered plants were fully mature (about 65 days old), the seeds of each plant of Col-0, ubp15-1 and ubp15-2 under watered and drought conditions were carefully collected. After the seeds were dried in an oven at 37°C for 1 week, the seed yield of each plant was counted on a micrometer balance. The results are shown in Figure 1b. The seed yield of ubp15-1 and ubp15-2 plants under drought conditions was significantly higher than that of Col-0 plants (63% and 77% higher, respectively), although the seed yield of both plants under watered control was slightly lower than that of Col-0 plants.

Regarding seed size, the seeds of the marked flowers were collected and photographed under a stereo microscope. The length and width of the seeds were counted using Digmizer software. The results showed that the length and width of the seeds of ubp15-1 and ubp15-2 plants under normal watering control conditions were significantly smaller than those of Col-0 plants, which was consistent with previous literature reports. Under drought conditions, the seed size of Col-0 plants was significantly smaller than that of Col-0 plants under watering control conditions, and the length and width of the seeds also decreased significantly, indicating that drought can induce Arabidopsis to produce small seeds. However, there was no significant difference between the seeds produced by ubp15-1 and ubp15-2 plants under drought conditions and those produced under watering control conditions, and there was no difference in the statistical data of seed length and width (as shown in Figures d and e in Figure 1).

The above data indicate that knocking out the Arabidopsis ubp15 gene can effectively prevent the reduction of branch numbers, growth inhibition, smaller seeds and lower yield under drought conditions, thereby effectively increasing plant yields under drought conditions and helping to stabilize yields.

Therefore, the UBP15 gene can be knocked out/knocked down by gene editing, T-DNA insertion, chemical/physical radiation mutagenesis, or the expression of the UBP15 gene can be inhibited by gene silencing, transcriptional regulation, or the activity of the UBP15 protein can be inhibited by inhibitors, protein modification, and other technologies, so as to obtain Arabidopsis thaliana and other plant seeds or plants with UBP15 gene function knockout, low expression, and low protein activity, which can be used for planting in arid areas or environments to avoid the inhibition or reduction of growth, seed size, tillering/branch number, yield and other factors of Arabidopsis thaliana and other plants under drought conditions, and to obtain improved Arabidopsis thaliana and other plant germplasm with high-yield and stable yield genes under drought conditions.

Example 2 Monitoring of Arabidopsis UBP15 protein degradation under drought conditions

1. Obtaining UBP15 CDS fragments

The Arabidopsis thaliana UBP15 CDS sequence was amplified by PCR, RNA from 7-day-old Col-0 seedlings grown on MS medium was extracted using Tranzol (Beijing Quanshijin, ER501), and the first-strand cDNA was synthesized using a reverse transcription kit (Beijing Quanshijin, AH311-02) with the extracted RNA as a template.

Using the first-strand cDNA as template, UBP15 CDS was amplified using the UBP15 CDS amplification primer pair pCambia-UBP15 F: 5'-ATCTGGGGCCCA TAGGCCTGATGCTTGAACCAAGGGGAGCG-3' and pCambia-UBP15 R: 5'-ATCCCCGGGTACCGAGCTCGCTACCAGTAACTGTAAGTTCTATCC-3'. The PCR amplification system was (2×KOD onemix: 50 μL, pCambia-UBP15 F: 2 μL, pCambia-UBP15 R: 2 μL, first-strand cDNA: 2 μL, ddH ₂ O: 44 μL), and the PCR program was (98℃: 3 min; 98℃: 10 s, 53℃: 5 s, 68℃: 30 s, 34 cycles; 68℃: 3 min; 16℃: 10 min).

The PCR product was electrophoresed in a 0.8% agarose gel (120 V, 30 min), and the gel containing UBP15 CDS (2787 bp) was cut out and the UBP15 CDS fragment was recovered using an agarose gel recovery kit (Magen, DP209-03).

2. Construction of 35S::myc-UBP15 overexpression vector

pCAMBIA35s-4×Myc-MCS-3×FLAG (Fenghui, FH1029) was digested with EcoRI-HF (NEB, R3101) at 37°C overnight, and the resulting digested vector was purified and recovered using an agarose gel recovery kit (Magen, DP209-03).

The purified and recovered enzyme-digested pCAMBIA35s-4×Myc-MCS-3×FLAG and the recovered UBP15 CDS fragment were recombined using a seamless cloning kit (Clone Smarter, C5891-50). The recombinant product was transferred into Top10 colon sensory competent cells (Saihongrui, CC0102), and then cultured on LB solid plates containing 50 mg/L kanamycin at 37°C overnight. Single clones were selected the next day, and colony PCR amplification of pCambia-UBP15 F and pCambia-UBP15 R was performed using UBP15 CDS amplification primers.

In the colony PCR amplification, the PCR system (2× Taq mix: 5 μL; pCambia-UBP15 F: 0.2 μL; pCambia-UBP15 R: 0.2 μL; a small amount of monoclonal colony was picked with a toothpick; ddH ₂ O: 4.6 μL), PCR program (95°C: 10 min; 95°C: 30 s, 60°C: 30 s, 72°C: 3 min, 30 cycles; 72°C: 5 min; 16°C: 10 min);

For colonies that were positive for the PCR-amplified UBP15 fragment, plasmids were extracted (Magen, DP103-03) and sent to a sequencing company (Reboxinco) for sequencing, and the 35S::myc-UBP15 overexpression vector with completely correct sequencing was selected and confirmed.

3. Arabidopsis thaliana infection

The correctly sequenced 35S::myc-UBP15 overexpression vector was transformed into GV3101 Agrobacterium competent cells by electroporation, and then colony PCR was performed according to the above method to confirm the positive Agrobacterium (i.e., Agrobacterium containing the 35S::myc-UBP15 vector).

The Agrobacterium containing the 35S::myc-UBP15 vector was propagated in LB liquid medium containing 25 mg/L rifampicin and 50 mg/L kanamycin by shaking, and then propagated again by large-scale propagation, and the bacterial liquid was collected by centrifugation (5000 r/min, 5 min) during the period of _OD600 = 0.5-0.7;

Use the inflorescence infection solution to resuspend the bacterial solution collected by centrifugation (100mL of bacteria is resuspended with 30ml of infection solution). After infecting the vigorously flowering Arabidopsis Col-0 inflorescence with the Agrobacterium resuspension, place it in high humidity and dark conditions overnight. Take it out the next day and culture it normally; repeat the infection once more.

Among them, the system of 100mL of flower tinfoil impregnation liquid is: MS salt (i.e. (2,3-dioleoyl-propyl)-trimethylamine) 0.132g; Sucrose (i.e. sucrose) 5g; Silwet L-77 30μL; 6-benzylaminoadenine (i.e. 6-BA) with a concentration of 1mg/mL 6μL; 4-morpholineethanesulfonic acid (i.e. MES) 0.03g, pH=5.7.

IV. Screening of 35S::myc-UBP15 transgenic lines

After the infected seeds mature, they are collected and dried, and the seed surface is disinfected with seed surface disinfectant (20% 84 disinfectant, 0.1% polyethylene glycol octylphenyl ether, i.e. Triton X-100) for 10 minutes. After washing 6 times with sterile water, the seeds are treated at 4°C for 3 days.

Considering that the pCAMBIA35s-4×Myc-MCS-3×FLAG vector used in the construction of the 35S::myc-UBP15 overexpression vector can make the transgenic line acquire hygromycin resistance, the seeds were sown on MS medium containing 25 μg/mL hygromycin to screen positive 35S::myc-UBP15 transgenic plants (i.e., positive seedlings) with resistance;

5. Screening and identification of plants with high myc-UBP15 protein accumulation

When the positive seedlings are 30 days old, pick one leaf (about 50 mg) that is fully expanded at the same time, grind the leaf tissue with liquid nitrogen, add 100 μL of ice-cold RB buffer B protein extract, mix thoroughly, incubate on ice for 5 minutes, and centrifuge at 13000r/min and 4℃ for 10 minutes. Carefully take the supernatant to obtain the total protein of the plant (including myc-UBP15 protein).

After the concentration of total protein was determined using the Bradford protein quantification kit (Thermo, 23200), 100 μg of total protein was loaded onto SDS-PAGE gel, and the myc-UBP15 protein was detected using the immunoblotting hybridization method and MYC antibody (1:2000) and goat anti-mouse secondary antibody (1:2000) to screen for overexpression strains with high accumulation of myc-UBP15 protein. After two generations of reproduction, homozygous 35S::myc-UBP15 transgenic seeds that did not separate under hygromycin resistance selection were obtained.

Among them, RB buffer B protein extract is 50mM Tris-HCl buffer (pH7.8), 100mM NaCl, 0.1% Tween 20 (i.e. Tween 20) by volume, 10% glycerol (i.e. glycerol) by volume, 20mM β-mercaptoethanol (i.e. β-mercaptoethanol), 1×Coctail (phosphatase inhibitor), and 20μM MG132 (proteasome inhibitor).

VI. Drought response experiment of UBP15 protein level

Homozygous 35S::myc-UBP15 transgenic seeds were planted and cultured normally. After bolting, the resulting 35S::myc-UBP15 transgenic plants were divided into two batches. One batch was subjected to drought treatment without water, and the other batch was treated with normal watering as a control. After 10 days of water shortage, the 35S::myc-UBP15 transgenic plants wilted. At this time, leaves of the 35S::myc-UBP15 transgenic plants under control and drought treatment were taken. After total protein was extracted and quantified according to the above method, the accumulation of myc-UBP15 protein was detected by immunoblotting hybridization experiment, and Actin antibody was used as an internal reference control (Actin antibody dilution ratio 1:5000).

The results are shown in Figure 1c. The accumulation of myc-UBP15 protein under drought conditions is almost undetectable, while the accumulation of myc-UBP15 protein under watering is high, indicating that UBP15 is a drought-responsive gene and its protein level is downregulated in response to drought, thereby inducing drought avoidance response phenotypes such as smaller seeds, making the plant more adaptable.

Example 3: Comparison of UBP15 genes in different species

The amino acid sequence encoded by the Arabidopsis thaliana UBP15 gene (At1g17110) or rice UBP15 gene (LOC_Os02g14730) was used as the reference sequence, and the NCBI Blast tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) or the Phytozome Blast tool (https://phytozome-next.jgi.doe.gov/blast-search) was used to compare the homologous sequences of UBP15 in the corresponding species, including maize (Zm00001eb238660), wheat (Phytozome:Traes_6DS_D2658E3DE), soybean ( Glycine max ( Glycine max 15G248200), and soybean ( Glycine max 15G248200). ), millet (Seita.1G020500), cassava (Manes.15G100900), sorghum (Sobic.004G107200), rapeseed (NCBI: LOC106387600), banana (NCBI: LOC103978569), sweet potato (Iba_chr09dCG2510), oil palm (NCBI: LOC105040636), papaya (Phytozome: evm.TU.supercontig_77.86), cocoa (Phytozome: Thecc1EG032321t1), coffee (Phytozome: evm.model.Scaffold_770.361), potato (Phytozome: KY284_031222), tobacco (NCBI: LOC107782039), etc.

The above-mentioned UBP15 homologous sequences were stored in a txt file in fasta format, and DNAMAN software was used to compare the UBP15 homologous sequences in the txt file to see whether the sequences had the USP domain (Prosite number: PS00972/PS50235 or ProRule number: PRU01035) and the newly named CDU15 domain in the present invention. If the sequences had the above two key domains, they could be defined as the UBP15 gene. The specific results are shown in Figure 2.

Example 4 UBP15 characteristic domain CDU15 domain naming and characteristic sequence publication

By comparing the homologous protein sequences of UBP15 from different species, it was found that there is a very conserved domain at the C-terminus of the UBP15 gene, and its characteristic sequence is SE(D)WS(H)LFTSSDE(D)S(A)SFT(S)TEST(.) RDSFSV(T)V(I/A)DY, where the amino acids in parentheses are replaceable residues of the residue before the parentheses, and "." indicates a residue deletion. The domain containing this conserved sequence is named CDU15 (Conserved-Domain of UBP15) in the present invention, with specific reference to Figure 2.

Example 5 Obtaining Rice UBP15 Gene Editing Knockout Mutants

To further verify that UBP15 gene knockout can promote plant yield under drought, rice was selected as a monocot and crop representative for research. First, the rice UBP15 gene (LOC_Os02g14730) edited mutant was ordered from Biogle (http://biogle.cn/). The edited mutant was knocked out of the first exon of the UBP15 gene (LOC_Os02g14730) using CRISPR-Cas9 technology in the ZH11 rice variety background using the targeting sequence 5'-ATGGGTCTTAAAGAAGGTCCTGG-3', as shown in Figure 3a.

In order to obtain a homozygous edited ubp15 knockout mutant without T-DNA insertion and eliminate the interference of T-DNA, the hygromycin resistance gene primers 5'-GAAGTGCTTGACATTGGGGAGT-3' and 5'-AGATGTTGGCGACCTCGTATT-3' in T-DNA were first used to obtain a T-DNA-free strain, and then the DNA of these T-DNA-free strains and the UBP15 target site specific amplification primers 5'-AGCTTTGGCCTGAGAGATCG-3' and 5'-GGCAACTTGAGGAGTGGCAA-3' were used to amplify the UBP15 gene fragment containing the target site, and the fragment was sequenced using primer 5'-AGCTTTGGCCTGAGAGATCG-3' to screen and obtain a homozygous mutant of the ubp15 gene editing without transgenic components (i.e., GACC was deleted and ACCAA was inserted), as shown in Figure 3b.

Example 6 Drought treatment of rice ubp15 knockout mutant potted plants

One plant of the ubp15 gene-edited homozygous mutant without transgenic components identified in Example 5 (i.e., rice ubp15 gene-edited knockout mutant) and one plant of the wild-type Zhonghua ZH11 were planted in the same rice culture pot, with a total of 8 pots planted. They were cultured under 12 h light/12 h dark, 28 ° C conditions, and water and fertilizer were applied normally during the vegetative growth period before bolting.

Since the knockout of ubp15 gene did not significantly affect the heading time, after the homozygous mutant of ubp15 gene editing without transgenic components and wild-type Zhonghua ZH11 began to bolt, 4 of the pots were deprived of water and drought treatment. During the period when the rice wilted, each pot was sprayed with 100 ml of water with a sprinkler to keep the rice in a state of water shortage and drought without dying. The other 4 pots were used as the control group and were watered normally (no fertilizer was applied).

About 45 days after treatment, rice seeds matured. During the planting process, the overall phenotypes of the non-transgenic ubp15 gene-edited homozygous mutant plants and the wild-type Zhonghua ZH11 plants are shown in Figure 4a. Under normal watering, the non-transgenic ubp15 gene-edited homozygous mutant plants and the wild-type Zhonghua ZH11 plants grew equally, and there was no significant difference in the number of tillers between the two. However, under drought conditions, the number of effective tillers of the non-transgenic ubp15 gene-edited homozygous mutant plants (as shown by the white triangles in Figure 4a) was significantly higher than that of the wild-type Zhonghua ZH11 plants under drought conditions, and even higher than that of the non-transgenic ubp15 gene-edited homozygous mutant plants and the wild-type Zhonghua ZH11 plants under the control (as shown in Figure 4b), indicating that the UBP15 gene plays a conservative and key role in regulating the number of tillers/branches and growth and development of rice in response to drought.

This is consistent with the phenotypes of Arabidopsis ubp15-1 (SALK_018601) homozygous mutants and ubp15-2 (SALK_015611) homozygous mutants after drought, indicating that the UBP15 gene plays a conserved and key role in regulating tiller/branch number and growth development in plant response to drought.

After collecting individual seeds of the non-transgenic ubp15 gene-edited homozygous mutant plants and the wild-type Zhonghua ZH11 plants, the yield was statistically analyzed and it was found that compared with the watered control, the single millet yield of the wild-type Zhonghua ZH11 plants under drought conditions was significantly reduced, while the single millet yield of the non-transgenic ubp15 gene-edited homozygous mutant plants under drought conditions was significantly higher than that of the wild-type Zhonghua ZH11 plants under the same conditions. Moreover, the drought-induced yield decrease of the non-transgenic ubp15 gene-edited homozygous mutant plants (-62.6%) was significantly smaller than that of the wild-type ZH11 plants (-84%) (as shown in Figure 4c). This is also consistent with the yield phenotype of the Arabidopsis thaliana ubp15-1 (SALK_018601) homozygous mutant and ubp15-2 (SALK_015611) homozygous mutant under drought conditions, indicating that UBP15 gene knockout can significantly increase the yield of monocotyledonous and dicotyledonous plants.

Subsequently, the seed size phenotypes of the collected non-transgenic ubp15 gene-edited homozygous mutant plants and the wild-type Zhonghua ZH11 plants were compared. It was found that compared with the watered control, drought conditions significantly induced the wild-type Zhonghua ZH11 plants to produce smaller seeds, but drought conditions did not significantly induce the seeds of the non-transgenic ubp15 gene-edited homozygous mutant plants to become smaller. In addition, the seeds of the non-transgenic ubp15 gene-edited homozygous mutant plants were smaller than those of the wild-type Zhonghua ZH11 plants under the watered control, and the 100-grain weight statistics of each individual plant were also consistent with the observation results (as shown in Figure 4, Figure 4D and Figure 4E). It can be seen that the seed size of the rice non-transgenic ubp15 gene-edited homozygous mutant does not decrease with drought environment. This phenotype is consistent with the Arabidopsis thaliana ubp15-1 (SALK_018601) homozygous mutant and ubp15-2 (SALK_015611) homozygous mutant, indicating that the UBP15 gene plays a key role in regulating seed size in plant response to drought.

Example 7 Water-saving treatment of rice ubp15 knockout mutant in the field

The non-transgenic ubp15 gene-edited homozygous mutant seeds and wild-type Zhonghua ZH11 seeds were planted in paddy fields in Sanya, Hainan in November. The paddy fields were equipped with greenhouses to avoid the impact of rainfall on the water-saving experiment.

For water-saving treatment, after the paddy field is plowed and leveled, a 5m×5m highland is isolated in the middle of the field. Two layers of ridges are built around the highland, and a ditch is left in the middle of the ridge to avoid external water interference on the highland. The periphery of the highland is the irrigation area.

The seeds of the non-transgenic ubp15 gene-edited homozygous mutant and the wild-type Zhonghua ZH11 were planted alternately in the highland water-saving area and the peripheral irrigation area. Among them, 25 plants of the non-transgenic ubp15 gene-edited homozygous mutant and the wild-type Zhonghua ZH11 were planted in the highland water-saving area and the peripheral irrigation area, that is, 25 plants of the non-transgenic ubp15 gene-edited homozygous mutant and 25 plants of the wild-type Zhonghua ZH11 were planted in the highland water-saving area (referred to as the water-saving treatment), and 25 plants of the non-transgenic ubp15 gene-edited homozygous mutant and 25 plants of the wild-type Zhonghua ZH11 were planted in the peripheral irrigation area (referred to as the irrigation treatment).

In the early stage, the ridges were dug, and the peripheral irrigation area and the highland water-saving area were irrigated normally. In the early stage of bolting, the highland water-saving area was drained and water was cut off, while the irrigation area was irrigated normally until the ears matured and turned yellow. In the late stage of water-saving treatment, the soil in the highland water-saving area cracked and turned white.

The average phenotype lines were selected and photographed. The results are shown in Figure 5a. The wild-type Zhonghua ZH11 plants in the highland water-saving area grew weaker than the wild-type Zhonghua ZH11 plants in the peripheral irrigation area, with fewer tillers and yellowing plants. However, the non-transgenic ubp15 gene-edited homozygous mutant plants in the highland water-saving area grew better than the non-transgenic ubp15 gene-edited homozygous mutant plants in the peripheral irrigation area, and the number of tillers did not decrease significantly, and even increased slightly.

Similarly, statistics on the number of effective tillers found that water-saving treatment led to a decrease in the number of tillers in wild-type ZH11 plants, but this inhibitory effect did not occur in the homozygous mutant plants of the ubp15 gene edited without transgenic components (Figure 5b), which is consistent with the trend of the potted results in Example 6, indicating that the UBP15 gene plays a key role in the branching response of rice to lack of regulation.

Subsequent statistics on the seed yield of individual plants showed that water-saving treatment significantly inhibited the yield of wild-type Zhonghua ZH11 plants (-40.0%), while the yield of the homozygous mutant plants of the ubp15 gene editing without transgenic components did not decrease under water-saving treatment compared with normal irrigation, but instead showed an upward trend (+12.3%) (Figures c and d in Figure 5), which is consistent with the statistics of the number of tillers (Figure b in Figure 5). At the same time, the single-plant yield of the homozygous mutant plants of the ubp15 gene editing without transgenic components under water-saving conditions was 43% higher than that of the wild-type Zhonghua ZH11 plants, which is consistent with the performance of the potted experiment in Example 6 (Figure c in Figure 4). This result further shows that the mutant ubp15 gene can effectively increase the yield of rice under drought or water-saving conditions, and gene editing of the mutant UBP15 gene is a feasible solution for creating water-saving rice.

Therefore, the UBP15 gene can be mutated by gene editing, T-DNA insertion, chemical/physical radiation mutagenesis, or the expression of the UBP15 gene can be inhibited by gene silencing, transcriptional regulation, or the activity of the UBP15 protein can be inhibited by inhibitors, protein modification, and other technologies, so that rice seeds that do not express or express less ubp15 gene (i.e., rice seeds with UBP15 gene mutation or gene editing) can be used for planting in arid areas or environments to avoid the inhibition or reduction of factors such as rice growth, seed size, number of tillers/branches, and yield under drought conditions, so as to obtain improved rice with high-yield and stable yield genes under drought conditions, thereby increasing rice yield.

In summary, the conserved homologous protein UBP15 in angiosperms can be used to avoid plant growth inhibition, seed size reduction, reduced tillering, and reduced yield under drought conditions;

The conserved homologous proteins UBP15 in angiosperms include Arabidopsis UBP15 (AT1G17110), rice UBP15 (Os02g0244300/LOC_Os02g14730) and homologous proteins in other species;

The amino acid sequence of Arabidopsis thaliana UBP15 is shown in SEQ ID NO: 1;

The amino acid sequence of rice UBP15 is shown in SEQ ID NO: 2;

The conserved homologous protein UBP15 of angiosperms includes both USP domain (Prosite number: PS00972/PS50235 or ProRule number: PRU01035) and CDU15 domain, including but not limited to Arabidopsis thaliana UBP15 (At1g17110), rice UBP15 (LOC_Os02g14730), homologous proteins of other species or proteins encoded by other artificial genes;

Genes encoding both the characteristic amino acid domains USP and CDU15, including but not limited to the Arabidopsis thaliana UBP15 (At1g17110) gene, the rice UBP15 (LOC_Os02g14730) gene, homologous genes of other species or other artificial genes;

Among them, the gene sequence of Arabidopsis thaliana UBP15 is shown in SEQ ID NO: 3;

The gene sequence of rice UBP15 is shown in SEQ ID NO: 4;

The CDS sequence of the Arabidopsis thaliana UBP15 gene is shown in SEQ ID NO: 5;

The CDS sequence of the rice UBP15 gene is shown in SEQ ID NO: 6;

>USP domains are:

GL(I)V(X)NCGNSCYANAV(A)LQS(O)LT(M)C(X)TKPLV(X)A(I/V)Y(F/H)LLR(X)RS(L)HSR( K)S(X)C(S/Y)S(C/Y)G(X)K(R)D(N)WCLM(V)CELEQ(K/R)H(Y)V(A)M( S)M(T)LR(K)ES(X)GG(X)PL(V)SA(P)S(N)R(K)I(F)LS(L)H(Q/R)M( I /L)R(Q)S(N/G)IN(G)C(S/G)Q(RH)I(L/M)GD(X)GSQEDAHEFLRL(H)L(I)V(I)A (M)SMQS(G/A)I(X)CLE(D)R(G/A)L(Q)GGET(X)KV(I)D(E/N)P(X)R(S/I )L(Q)QE(D/Q)TTL(F)V(I)QH(Q)M(T/I)FGGR(Q)LR(KQ)SKVKCL(Q)R(N)CD(X) H(L/V)ESE(A)R(C)Y(H/S)EN(S)IMDLT(S)LEIY(X)GW(R)VE(Q)SLQ(E)DALTQFTR(X)PED( E)LDGE(D)NMYR(K)CS(G)R(S)CA(X)G(X)YVR(K/E)AR(Q)KE(Q)LS(C)I(V)H( Q)EA(V)PNILTI(V)VLKRFQ(K)E(X)GR(X)YGKINKCI(V)S(T/A) FPE(D)MLDMI(V)P(Y)FM(V)TR(G)T(A/S)G(D/A)DV(X)PPLYM(F)LYAVI(V)VHL(V)DT( E)LNASFSGHYI(V)S(A/T)YV(I)KDL(M)R(Q/H)GN(T)WY(X)RI(V)DDS(T)E(K)I(V) H(Q/K)P(X)VP(X)M(X)T(X)Q(R)VMS(T)EGAYM(I)LFYM(X)RS;

Note: The amino acids in parentheses are possible alternative amino acid residues before the parentheses, and "X" indicates that there are three or more alternative amino acid residues;

>The CDU15 domains are:

SE(D)WS(H)LFTSSDE(D)S(A)SFT(S)TEST(.)RDSFSV(T)V(I/A)DY;

Note: The amino acids in parentheses are possible replacement amino acid residues before the parentheses. "(.)" indicates that the "T" before the parentheses may be missing.

The full name of the CDU15 domain is "Conserved-Domain of UBP15", and its Chinese name is "UBP15 conserved domain";

The CDU15 domain is a characteristic sequence of the UBP15 protein or the product encoded by the UBP15 gene, and can be used to characterize UBP15 homologous proteins in different species.

The function of the conserved homologous protein UBP15 in angiosperms is to avoid plant growth inhibition, seed size reduction, reduced tillering/branching number, and reduced yield under drought conditions by reducing UBP15 protein levels, inhibiting UBP15 protein function, or knocking out/knocking down the UBP15 gene, thereby achieving stable yield.

The application of the conserved homologous protein UBP15 in angiosperms is to avoid plant growth inhibition, seed size reduction, reduced tiller/branch number and yield reduction under drought conditions by mutating or editing the UBP15 gene, thereby promoting high and stable yield of plants under drought conditions.

Furthermore, by means of UBP15 gene knockout, UBP15 gene knockdown, UBP15 protein level reduction or UBP15 protein function inhibition, the growth of angiosperms under drought conditions is inhibited, seeds become smaller, the number of tillers/branches is reduced, and the yield is reduced, thereby promoting high and stable yields of plants under drought conditions;

UBP15 gene knockout was performed using any of the following techniques:

Knockout of the UBP15 gene using T-DNA insertion;

Knock out the UBP15 gene using gene editing, such as CRISPR/Cas, ZFN, TALEN and other gene editing technologies;

Knock out the UBP15 gene by chemical mutagenesis, such as mutagenesis treatment with chemical mutagens such as EMS;

Knocking out the UBP15 gene by physical mutagenesis, such as space radiation, alpha radiation, beta radiation, neutrons and other particles, ultraviolet radiation, and microwave radiation;

The UBP15 gene knockdown was performed using any of the following techniques:

Knock down the UBP15 gene using gene editing, such as CRISPR/Cas, ZFN, TALEN and other gene editing technologies;

Knock down the UBP15 gene using gene silencing technology, including RNAi interference technology and other gene silencing technologies;

Knockdown of UBP15 gene by promoter changes, such as gene editing promoter sequence, T-DNA insertion promoter sequence, etc.

Knockdown of UBP15 gene by modifying key factors that regulate UBP15 gene expression;

Knock down the UBP15 gene using chemical reagents or physical factors;

The reduction of UBP15 protein level or inhibition of UBP15 protein function is achieved by any of the following techniques:

Reduction of UBP15 protein levels by effective molecular treatment;

The UBP15 protein level is reduced by effective environmental treatments such as drought;

Reduction of UBP15 protein levels caused by other protein manipulation techniques;

Inhibition of UBP15 function caused by protein modification, molecular interaction, inhibitors, etc.

Drought conditions include any of the following:

Cut off water in the late vegetative growth stage or early reproductive growth stage of plants until the plants completely dry up and die;

Or, when the plant is deprived of water in the late vegetative growth stage or early reproductive growth stage, the plant will wilt as a sign;

Alternatively, under water-saving treatment in the late vegetative growth stage or early reproductive growth stage of plants, the phenotype may be marked by a significant decrease in moisture content, such as cracking of the field soil.

Other parts not described in detail are all prior art. Although the above embodiment has made a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. A person skilled in the art can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims (10)

1. The protein is used for effectively improving the yield of the arabidopsis/rice under the water-saving or drought condition, and is characterized by being an arabidopsis/rice conserved homologous protein UBP15 for preventing the growth inhibition, seed reduction, tillering reduction and yield reduction of the arabidopsis/rice under the drought condition;

arabidopsis/rice conserved homologous protein UBP15 comprises Arabidopsis UBP15 and rice UBP15;

Wherein the amino acid sequence of the Arabidopsis UBP15 is shown as SEQ ID NO:1 is shown in the specification;

The amino acid sequence of rice UBP15 is shown as SEQ ID NO: 2.

2. The protein effective to increase yield of arabidopsis/rice under water conservation or drought conditions of claim 1, wherein said drought conditions comprise any one of:

Under the treatment of water interruption in the late vegetative growth stage or early reproductive growth stage of the arabidopsis thaliana/rice, until the arabidopsis thaliana/rice is completely dead;

Or, under the treatment of water interruption in the late vegetative growth stage or early reproductive growth stage of the arabidopsis thaliana/rice, taking the arabidopsis thaliana/rice wilting as a sign;

or, under the water-saving treatment of the late vegetative growth or early reproductive growth of the arabidopsis/rice, the phenotype of remarkably reduced water content is taken as a mark.

3. Use of a gene encoding the protein of claim 1 or 2 effective to increase yield of arabidopsis/rice under water-saving or drought conditions, characterized in that the gene encoding the protein of claim 1 or 2 effective to increase yield of arabidopsis/rice under water-saving or drought conditions is UBP15 gene;

The application is the application of UBP15 gene in arabidopsis/rice drought response regulation and control growth, seed size, tillering/branching number and yield.

4. The use according to claim 3, wherein the use is to promote high and stable yield of arabidopsis/rice under drought conditions by inhibiting growth, seed size reduction, tillering/branching number reduction and yield reduction of arabidopsis/rice under drought conditions by means of UBP15 gene knockout, UBP15 protein level reduction or UBP15 protein function inhibition.

5. The use of claim 4, wherein the UBP15 gene knockout employs any one of the following techniques:

Knocking out UBP15 gene by using T-DNA insertion;

knocking out UBP15 gene by utilizing gene editing;

knocking out the UBP15 gene by chemical mutagenesis;

the UBP15 gene was knocked out by physical mutagenesis.

6. The use of claim 4, wherein UBP15 gene knockdown is by any of the following techniques:

knocking down UBP15 gene by gene editing;

knocking down the UBP15 gene by using a gene silencing technique;

knocking down UBP15 gene by promoter variation;

Modification of UBP15 gene expression key factors by regulating UBP15 gene expression key factors leads to knocking down of UBP15 genes;

the UBP15 gene is knocked down using chemical agents or physical factors.

7. The use according to claim 4, wherein said reduced UBP15 protein level or inhibition of UBP15 protein function employs any of the following techniques:

Reduced UBP15 protein levels caused by effective molecular treatment;

reduced UBP15 protein levels caused by effective environmental treatment;

reduced UBP15 protein levels by other protein manipulation techniques;

inhibition of UBP15 function by protein modification, molecular interactions, inhibitor effects are employed.

8. The use of claim 4, wherein the drought conditions include any of:

Under the treatment of water interruption in the late vegetative growth stage or early reproductive growth stage of the arabidopsis thaliana/rice, until the arabidopsis thaliana/rice is completely dead;

Or, under the treatment of water interruption in the late vegetative growth stage or early reproductive growth stage of the arabidopsis thaliana/rice, taking the arabidopsis thaliana/rice wilting as a sign;

or, under the water-saving treatment of the late vegetative growth or early reproductive growth of the arabidopsis/rice, the phenotype of remarkably reduced water content is taken as a mark.

9. The use according to any one of claims 3-8, wherein the use is to promote high and stable yield of arabidopsis/rice under drought conditions by avoiding inhibition of growth, seed reduction, tillering/branching number reduction and yield reduction of arabidopsis/rice under drought conditions by knocking out/knocking down UBP15 gene.

10. The use of claim 9, wherein said knockdown/knockdown of UBP15 gene is by mutation of UBP15 gene by techniques including gene editing, T-DNA insertion disruption, chemical/physical radiation mutagenesis, or by inhibition of UBP15 gene expression by techniques of gene silencing, transcriptional control, or by inhibition of UBP15 protein activity by techniques of inhibitor, protein modification.