CN106701756A - Application of microRNA172 to regulation of plant height, ear type and leaf size of rice - Google Patents

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to the application of microRNA172 in regulating rice plant height, panicle type and leaf size. The invention also provides the application method of the miR172 antagonist MIM172 gene fragment in rice genetic improvement. miR172b is the nucleotide sequence shown in SEQ ID NO:1; pri-miR172b is the nucleotide sequence shown in SEQ ID NO:2. Overexpression of pri-miR172b resulted in smaller rice leaves, less panicle branches and fewer grains per panicle. The antagonist MIM172 is the nucleotide sequence shown in SEQ ID NO: 3, and reducing the expression level of miR172 by the antagonist MIM172 will lead to shorter rice plant height, larger leaves, and increased panicle branches and panicle grains. These results indicate that miR172 can regulate the plant architecture of rice, which has important application value.

Description

Application of microRNA172 in regulating rice plant height, panicle shape and leaf size

technical field

The invention relates to the technical field of plant genetic engineering. Specifically related to a microRNA172b (hereinafter abbreviated as miR172b) and its precursor gene pri-microRNA172b (hereinafter abbreviated as pri-miR172b), microRNA172 (hereinafter abbreviated as miR172) antagonist MIM172 in the regulation of rice plant height, panicle type and leaf size Applications. Increasing the expression of miR172b through genetic engineering technology will lead to the reduction of rice panicle branches, the number of panicle grains, the leaves become shorter, and the leaves become narrower; while the use of antagonist MIM172 (this antagonist is an artificially designed and synthesized 24bp long gene fragments, which can be used to reduce the expression of miR172), will lead to shorter plant height, more panicle branches, more panicle grains, longer leaves, and wider leaves. These results indicate that by manipulating the expression of miR172 Quantity can provide a method for improving the plant type of rice.

Background technique

With the continuous growth of the global population and changes in human consumption patterns, the global food crisis is becoming increasingly severe. Rice is one of the most important food crops in the world. More than half of the population in my country takes rice as the staple food. Therefore, increasing rice production is of great strategic significance to ensure national food security. In the last century, due to dwarf breeding and the use of heterosis, the yield of rice has undergone two qualitative leaps. However, in recent decades, the yield of rice has stagnated for a long time, and it is increasingly difficult to meet the needs of social and economic development. Therefore, new technologies and genetic resources are urgently needed to improve important agronomic traits such as yield, resistance, and quality of rice.

The yield of rice is closely related to its plant type, and the breeding model with the ideal plant type as the improvement goal has been highly valued in agricultural production. The plant architecture of rice mainly includes plant height, tiller, panicle type, root morphology, and the size and angle of organs, etc. (Wang and Li, Molecular basis of plant architecture. Annu Rev Plant Biol, 2008, 59:253-279). Among them, plant height, tillering, panicle shape and grain weight have long been important target traits for genetic improvement. Plant height is a trait closely related to rice yield, photosynthesis and lodging resistance. In the last century, the first "green revolution" aimed at reducing plant height greatly increased the yield of global crops. With the development of genetics and molecular biology, it has been clarified that hormones, especially gibberellin and brassinosterol, play a key role in regulating plant height (Wang and Li, Molecular basis of plant architecture.Annu Rev Plant Biol, 2008 , 59:253-279).

The panicle of rice is the purpose organ harvested in agricultural production, and its shape and composition directly determine the yield of rice. The panicle of rice is a kind of panicle, which is developed from the inflorescence meristem, which can generally produce primary branches and secondary branches, and in a few cases can also produce higher-level branches, which will further develop It becomes a spikelet and then produces a flower, and after flowering and fertilization of the flower organ, rice will be formed. Different from Arabidopsis and other plants, rice panicle development is determined by a series of meristem fate transitions. Under suitable internal and external environmental conditions, rice grows from vegetative growth to reproductive growth. At the same time, the apical meristem will also transform into inflorescence meristem, and the branch meristem will be generated from the inflorescence meristem. After a branch meristem, the inflorescence meristem will degenerate. The primary branch meristem will produce a higher-level secondary branch meristem, which will also directly differentiate into the spikelet meristem, and the secondary branch meristem will repeat the developmental pattern of the primary branch meristem , and eventually branch meristems are transformed into spikelet meristems. The spikelet meristem produces the floral meristem, which in turn produces the differentiation of floral organs. The transition time of different meristems largely determines the panicle type of rice (Kyozuka et al., Control ofgrass inflorescence form by the fine-tuning of meristem phase change.Curr Opin Plant Biol,2014,17:110-115 ). If the inflorescence meristem is maintained for a longer period of time, more primary branches will be produced; and if the differentiation of the spikelet meristem is delayed, more secondary branches or even tertiary branches may be produced, resulting in More grains per spike. In view of the importance of panicle type in agricultural production, scientists have discovered a large number of genes controlling the development of panicle type, and some genes have been applied in breeding practice (Zhang and Yuan, Molecular control of grass inflorescence development. Annu Rev Plant Biol, 2014, 65:553-578).

The growth and development of plants requires a lot of energy, and photosynthesis is the main source of energy for plants. Leaf is the most important organ for photosynthesis in plants. Its growth and development not only determines the plant shape of plants, but also determines the ability of plants to carry out photosynthesis and assimilate carbohydrates. Rice leaves include blades and leaf sheaths, and leaf pillows are formed at the junction of the two, and leaf pillows often differentiate into auricles and ligules. The leaf is developed from the apical meristem in the vegetative growth phase, and the polar transport of auxin will lead to the formation of a local zone of maximum auxin concentration at the edge of the meristem, in which the meristem The specific expression gene OSH1 will be inhibited, so that the cells in this area will eventually differentiate into leaf primordia cells and start the leaf development program. With the development of leaf primordium, the polarity of apical base, ventral dorsal and middle margin will be gradually formed, thus determining the spatial pattern of leaves. The final size of the leaf is determined by the combination of cell division and cell extension.

microRNA (miRNA) is a class of endogenous non-coding RNA molecules with a length of about 20-24 nucleotides. A large number of miRNA-coding genes have been found in almost all studied higher organisms. miRNA often controls the activity of target gene mRNA by regulating the stability of mRNA or regulating the process of protein translation, thereby regulating various life activities including growth, development, metabolism, and response to the environment. Because of its powerful biological functions and complex regulatory mechanisms, miRNA-related research has become one of the hot areas of life science today, and may play an important role in the genetic improvement of crops.

Many miRNAs are encoded in rice, but most of their biological functions are still unknown, and more work is urgently needed to elucidate the role of these small RNAs and their utilization value in rice genetic improvement. In addition, it is very difficult to obtain loss-of-function mutants of specific miRNAs, given that the same miRNA is often encoded by multiple precursor genes. It has been reported that a technology called target mimicry can effectively reduce the expression of miRNA in plants (Franco-Zorrilla et al., Target mimicry provides a new mechanism for regulation of microRNA activity.Nat Genet, 2007,39:1033- 1037). The technology works by designing a simulated target RNA that partially matches the target miRNA, which can bind to the miRNA but cannot be cut by the miRNA. Overexpression of this simulated target RNA in plants can bind to the target miRNA, preventing the miRNA from acting on the real target gene in the plant; meanwhile, the simulated target RNA can also cause the target miRNA to be degraded through an unknown mechanism. Although this technique can be used to interfere with miRNA activity, it has rarely been used for genetic improvement of rice.

The present invention studies the function of miRNAs in rice by means of plant genetic engineering, excavates miRNAs that may be used to change the plant type of rice in agricultural production, so as to provide new genetic resources for rice breeding; and utilizes target mimicry technology to interfere with these miRNA activity to improve rice agronomic traits.

Contents of the invention

The purpose of the present invention is to provide an application of miR172b, precursor gene pri-miR172b, and miR172 antagonist MIM172 in regulating rice plant type. Another object of the present invention is to provide a vector for overexpressing miR172b and MIM172, and the application of the vector in transgenic rice. At the same time, the invention also provides a method for changing the rice plant type by regulating the expression level of miR172. miR172b has the nucleotide sequence shown in SEQ ID NO: 1, and also includes gene sequences with more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 1, and also includes Mutant alleles or derivatives produced by one or more bases. The present invention also includes the precursor gene pri-miR172b encoding miR172b, the pri-miR172b has the nucleotide sequence shown in SEQ ID NO: 2, and has 90% of the nucleotide sequence shown in SEQ ID NO: 2 The above homologous gene sequences also include mutant alleles or their derivatives produced by insertion, substitution or deletion of one or more bases. Simultaneously, the present invention also includes the antagonist MIM172 of miR172, the antagonist MIM172 has the nucleotide sequence shown in SEQ ID NO: 3, and contains the DNA fragment of MIM172, and/or contains the expression vector of MIM172, and with SEQ ID NO:3 Derivatives with no more than 6 base differences in the nucleotide sequence shown in ID NO:3.

By increasing the expression of miR172b in the present invention, it will lead to the reduction of rice panicle branches, the number of panicle grains, the leaves become shorter, and the leaves become narrower; and the antagonist MIM172 is used to reduce the expression of miR172, which will lead to a change in rice plant height. These results indicated that manipulating the expression of miR172 could provide a new breeding method for improving the agronomic traits of rice. miR172b and its precursor gene pri-miR172b can be used to fuse with other regulatory elements, such as constitutive promoters or organ-specific promoters, to construct gene expression vectors; it can also be used by transgenic technology, antisense RNA, RNAi, zinc finger nuclease ( Zinc-finger nucleases, ZFN), transcription activator-like effector nucleases (transcription activator-like effector nucleases, TALEN) and CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 and other technologies to manipulate them to regulate plant architecture; The antagonist MIM172 can be used to fuse with other regulatory elements, such as constitutive promoters or organ-specific promoters, to construct gene expression vectors, so that the plant type of plants can be changed by means of genetic engineering.

Realize the concrete technical scheme of the present invention as follows:

1. According to the results of rice genome annotation, a DNA fragment containing the coding miR172b precursor gene pri-miR172b was isolated from rice. The specific isolation method is described in detail in Example 1.

2. The above-mentioned DNA fragment containing pri-miR172b was connected to the pCAMBIA1301S vector to construct an overexpression vector of miR172b. We named the vector 35S:miR172bOE; the specific construction method is described in detail in Example 2.

3. Using the method of target mimciry (Franco-Zorrilla et al., Target mimicry provides a new mechanism forregulation of microRNA activity. Nat Genet, 2007, 39: 1033-1037), design an artificial antagonist MIM172 that can interfere with miR172 activity. The nucleotide sequence is shown in SEQ ID NO: 3; and by the method of overlap extension PCR, MIM172 is fused to the non-coding protein gene AtIPS1 from Arabidopsis thaliana in order to construct an expression vector for genetic transformation; specific method It is described in detail in Example 3.

4. Connect the AtIPS1 fused with the antagonist MIM172 to the pCAMBIA1301S vector to construct the vector 35S:MIM172 overexpressing the antagonist MIM172. The specific method is described in detail in Example 4.

5. Using the optimized Agrobacterium-mediated transgenic method (Lin and Zhang, Optimizing the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell Rep, 2005, 23:540-548), the expression vector 35S:miR172bOE and 35S : MIM172 was introduced into the rice recipient Zhonghua 11 (Institute of Crop Science, Chinese Academy of Agricultural Sciences) to obtain transformed plants; the specific method is described in detail in Example 5.

6. Analyzing and identifying positive transgenic plants by means of PCR; and identifying the genotype and phenotype of the transgenic plants in the T1 generation, performing co-segregation detection, and performing statistical analysis on related phenotypes; the specific method is described in detail in Example 6 .

7. Utilize the method of stem-loop RT-PCR (Shen et al., Global expression profiling of rice microRNAs by one-tube stem-loop reverse transcription quantitative PCR revealed important roles of microRNAs in abiotic stress responses. Mol Genet Genomics, 2010, 284: 477-488) to analyze the expression level of miR172 in the transgenic plants; the specific method is described in detail in Example 7.

8. Analyze whether MIM172 antagonizes the effect of overexpression of miR172 by hybridization; the specific method is described in detail in Example 8.

Compared with prior art, advantage of the present invention is as follows:

1. The present invention found that overexpression of miR172b can reduce the length and width of leaves;

2. The present invention uses the antagonist MIM172 to regulate the expression of miR172 in rice for the first time;

3. The present invention found that using the antagonist MIM172 to reduce the expression level of miR172 in rice can lead to shorter rice plant height, increased panicle branches and spike grains, and larger leaves, and changes in these traits, especially panicle traits The change is in line with the needs of rice genetic improvement;

4. The present invention provides a method for the genetic improvement of rice plant type and leaves by regulating the activity of miR172.

Description of drawings

Sequence Listing SEQ ID NO: 1 is the nucleotide sequence of miR172b.

Sequence Listing SEQ ID NO: 2 is the nucleotide sequence of the precursor pri-miR172b of miR172b, and its 208th-228th nucleotide sequence encodes miR172b.

Sequence Listing SEQ ID NO: 3 is the nucleotide sequence of the artificially designed miR172 antagonist MIM172.

Figure 1: RNA secondary structure of the precursor gene pri-miR172b of miR172b. Explanation of reference signs:

The secondary structure is calculated according to the minimum free energy using the public software package ViennaRNA Package (http://www.tbi.univie.ac.at/RNA/), and the part shown in red letters is the sequence of miR172b.

Figure 2: Schematic diagram of the construction process of the expression vector 35S:miR172bOE. Explanation of reference signs:

Panel A in Figure 2: a schematic diagram of a DNA fragment containing pri-miR172b inserted into the expression vector 35S:miR172bOE.

Panel B in Fig. 2: Schematic diagram of the structure of the empty vector pCAMBIA1301S used to construct the expression vector 35S:miR172bOE. The present invention inserts the fragment shown in Figure A in Figure 2 through KpnI and BamHI double digestion into the KpnI and BamHI restriction sites of vector pCAMBIA1301S shown in Figure 2 in Figure 2 to form an expression vector for transformation 35S:miR172bOE .

Figure 3: Map of the expression vector 35S:miR172bOE constructed in the present invention.

Figure: 4: Schematic diagram of nucleotide pairing between artificial antagonist MIM172 and miR172b. Explanation of reference signs:

The red letter A indicates the 10th nucleotide of miR172b; the blue letter U indicates the 11th nucleotide of miR172b; the purple four letters indicate the small ring formed by the nucleotide mismatch with miR172 in MIM172 This structure is the key to the function of MIM172.

Figure 5: Schematic diagram of the process of fusing the antagonist MIM172 into the Arabidopsis AtIPS1 gene by the overlap extension PCR method. Explanation of reference signs:

Red, blue, green, and purple fragments represent regions in AtIPS1 used for primer design, while orange fragments represent the antagonist MIM172. When synthesizing primers MIM172F and MIM172R, the sequence of MIM172 was directly fused to the sequence of AtIIPS1.

Figure 6: Schematic diagram of the construction process of the expression vector 35S:MIM172. Explanation of reference signs:

Panel A in Figure 6: a schematic diagram of a DNA fragment containing antagonist MIM172 inserted into the expression vector 35S:MIM172.

Panel B in Figure 6: a schematic structural view of the empty vector pCAMBIA1301S used to construct the expression vector 35S:MIM172.

The present invention inserts the fragment shown in Figure A in Figure 6 through KpnI and BamHI double digestion into the middle of the KpnI and BamHI restriction sites of vector pCAMBIA1301S shown in Figure B in Figure 6 to form an expression vector for transformation 35S:MIM172 .

Figure 7: Map of the expression vector 35S:MIM172 constructed by the present invention.

Figure 8: Phenotype comparison between wild type Zhonghua 11 (WT) and 35S:miR172bOE (miR172OE) transgenic plants. Explanation of reference signs:

Panel A in Fig. 8: comparison of plant height and tiller morphology between wild-type Zhonghua 11 (WT) and 35S:miR172bOE (miR172OE) transgenic plants.

Panel B in Figure 8: comparison of panicle types between wild-type Zhonghua 11 (WT) and 35S:miR172bOE (miR172OE) transgenic plants.

Panel C in Figure 8: It is the primary branch morphology of wild-type Zhonghua 11 (WT) and 35S:miR172bOE (miR172OE) transgenic plants

Compare.

Figure 9: Phenotype comparison between wild type Zhonghua 11 (WT) and 35S:MIM172 (MIM172) transgenic plants. Explanation of reference signs:

Panel A in Fig. 9 is a comparison of plant height and tiller morphology between wild-type Zhonghua 11 (WT) and 35S:MIM172 (MIM172) transgenic plants.

Panel B in Figure 9: comparison of panicle types between wild-type Zhonghua 11 (WT) and 35S:MIM172 (MIM172) transgenic plants.

Panel C in Figure 9: is a branch morphology comparison between wild-type Zhonghua 11 (WT) and 35S:MIM172 (MIM172) transgenic plants.

Figure 10: Comparison of flag leaf morphology of wild-type Zhonghua 11 (WT), 35S:miR172bOE (miR172OE) and 35S:MIM172 (MIM172) transgenic plants.

Figure 11 : Detection of miR172b expression levels in wild-type Zhonghua 11 (WT) and 35S:miR172bOE (miR172OE) transgenic plants by real-time quantitative PCR. Data are shown as mean ± standard error of 3 replicates.

Figure 12: Real-time quantitative PCR was used to detect the expression level of miR172b in wild-type Zhonghua 11 (WT) and 35S:MIM172 (MIM172) transgenic plants. Data are shown as mean ± standard error of 3 replicates.

Figure 13: Antagonism of miR172 by MIM172. Explanation of reference signs:

A in Figure 13: comparison of panicle types of wild-type Zhonghua 11 (WT), 35S:miR172bOE (miR172OE) transgenic plants, 35S:MIM172 (MIM172) transgenic plants, and miR172OE and MIM172 hybrids.

B in Figure 13: the primary branch per panicle and the secondary branch per panicle of wild-type Zhonghua 11 (WT), 35S:miR172bOE(miR172OE) transgenic plant, 35S:MIM172(MIM172) transgenic plant, and miR172OE and MIM172 hybrids Statistics on the number of stalks and spikelets per panicle. Data shown are mean ± standard error from 10 individual plants.

detailed description

Example 1: Isolation of DNA fragments comprising the precursor gene pri-miR172b of miR172b

Using Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/) and miRBase (http://www.mirbase.org/) two websites to search, get miR172b (the gene accession number of miRBase database is MIMAT0001070 ) and the nucleotide sequence of its precursor gene pri-miR172b (gene accession number of miRBase database is MI0001140). The sequence of miR172b mature body is shown in SEQ ID NO:1, the sequence of its precursor gene pri-miR172b is shown in SEQ ID NO:2, and its secondary structure is shown in FIG. 1 .

According to the analysis results of the database sequence, in order to amplify the gene fragment of pri-miR172b, the following primers were designed:

MiR172bOEF (forward primer): 5'- GGTACC CAGTAGAGAGTGTGATGCCGCAGCT-3' (the underlined sequence is the KpnI recognition site);

MiR172bOER (reverse primer): 5'- GGATCC GCGGCGTTGGTACAATTAAGCTGATG-3' (the underlined sequence is BamHI recognition site).

Total DNA was extracted from leaves of rice variety Zhonghua 11 (Institute of Crop Science, Chinese Academy of Agricultural Sciences). The DNA extraction method was the CTAB method (Zhang et al., Genetic diversity and differentiation of indica an japonica rice detected by RFLP analysis. TheorAppl Genet, 1992, 83:495-499). Using the total leaf DNA of Zhonghua 11 as a template, PCR amplification was performed with primers MiR172bOEF and MiR172bOER. The total volume of the PCR reaction is 20 μl, including 100 ng of DNA template, 2 μl of 10xPCR buffer, 2 μl of 10 mM dNTP, 0.3 μl of 10mM primers MiR172bOEF and MiR172bOER, 0.2 μl of rTaq enzyme, and adding deionized water to 20 μl (used PCR buffer, dNTP, rTaq enzyme etc. were purchased from Treasure Bioengineering Dalian Co., Ltd.). The PCR reaction conditions are as follows: ① 94°C for 4 min, ② 94°C for 40 s, ③ 58°C for 40 s, ④ 72°C for 1 min, ⑤ cycles from ② to ④ 38 times, ⑥ 72°C for 7 min, and ⑦ storage at 4°C. The PCR product was detected by electrophoresis on 1% (mass/volume) TBE agarose gel, and the DNA fragment with a length of 317bp (the target DNA segment of 305bp plus the additional two restriction enzyme sites 12bp on the primer) was recovered . The recovered PCR product was ligated to the T-A cloning vector pGEMT-vector using T4 DNA ligase (T4 DNA ligase and vector pGEMT-vector were both purchased from Promega (Beijing) Biotechnology Co., Ltd., namely Promega, USA). The ligation product was introduced into Escherichia coli DH10B (purchased from Promega Company) by electroporation (the electrotransformer is a product of eppendorf, and the voltage used in the present invention is 1800V, and the specific operation refers to the instruction manual of the instrument), and 400 μl LB medium was added for recovery 45min, spread on LA medium plate containing ampicillin, 5-bromo-4-chloro-3-indole-β-D-galactose and isopropyl-β-D-thiogalactoside, 37℃ Incubation in an incubator for 14-16 hours (LA and LB formula reference: Sam Brook, "Molecular Cloning Experiment Guide" third edition, Science Press, 2002). Pick the single clone that appears blue, expand the culture and extract the plasmid, screen the positive clones by KpnI and BamHI double enzyme digestion, and use the T7 and SP6 primers on the vector to verify the inserted PCR product by sequencing. The PCR product contains the sequence of pri-miR172b shown in SEQ ID NO:2. The recombinant plasmid containing the PCR product was named plasmid TA-miR172b.

Embodiment 2: Construction of the overexpression vector of miR172b

KpnI and BamHI were used to double-digest plasmid TA-miR172b (the KpnI and BamHI were purchased from Bao Biological Engineering Dalian Co., Ltd., and the specific usage and dosage refer to the instructions of the corresponding products of the company. The plasmid TA-miR172b is from Example 1, as shown in the figure 2), the DNA fragment of 317bp containing pri-miR172b was recovered, and then combined with the vector plasmid pCAMBIA1301S [this vector was publicly reported: Zhou et al., Over-expression of aspartate through KpnI and BamHI double digestion aminotransferase genes in riceresulted in altered nitrogen metabolism and increased amino acid content in seeds. Theor Appl Genet, 2009, 118:1381-1390; its basic skeleton originated from the Australian CAMBIA laboratory (http://www.cambia.org/daisy/ cambia/materials/overview.html) pCAMBIA1301, by adding 35S promoter to realize the expression regulation of the transformed gene; its structure is shown in Figure B in Figure 2] Utilize T4DNA ligase (purchased from Promega Company, specific usage and dosage Refer to the manual of the company's product) to connect. The ligation product was introduced into Escherichia coli DH10B (purchased from Promega Company) by electroporation (the electrotransformer is a product of eppendorf, and the voltage used in the present invention is 1800V, and the specific operation refers to the instruction manual of the instrument), and 400 μl LB medium was added for recovery 45min, spread on the LA medium plate containing 50mg/L kanamycin, incubate at 37°C for 14-16h (LA and LB formula reference: Sam Brook, "Molecular Cloning Experiment Guide" third edition, Science Press, 2002). Single clones were picked, expanded and cultured, and plasmids were extracted. Positive clones were screened by KpnI and BamHI double enzyme digestion, and the resulting expression vector was named 35S:miR172bOE, and its structure is shown in Figure 3.

Example 3: Design of antagonist MIM172

According to published literature (Franco-Zorrilla et al., Target mimicry provides a new mechanism for regulation of microRNA activity. Nat Genet, 2007, 39:1033-1037), the method using target mimicry can interfere with the activity of plant miRNA, thus An antagonist MIM172 sequence that can antagonize the activity of miR172 is designed, and the sequence features are shown in SEQ ID NO:3. MIM172 is 24 nucleotides in length, 3 nucleotides more than miR172 of 21 nucleotides. The extra 3 nucleotides form a small looped protrusion. Because the mRNA regulated by miRNA in plants is generally cut from the middle position matching the 10th and 11th bases of miRNA (Schwab et al., Specific effects of microRNAs on the plant transcriptome. Dev Cell, 2005, 8: 517-527), therefore, the small ring-shaped protrusion formed by the three nucleotides in the antagonist MIM172 destroys the shearing effect of miR172, which is the key to the function of MIM172. miR172 in rice is encoded by four precursor genes (pri-miR172a, pri-miR172b, pri-miR172c and pri-miR172d), and the resulting four miR172 mature sequences are highly consistent, with the most The difference between the head and the tail is no more than 2 bases, so the MIM172 designed by the present invention can antagonize all forms of miR172 in rice, and its matching with miR172b is shown in FIG. 4 .

The applicant further connects the sequence of MIM172 by the method of overlap extension PCR (overlap extension PCR method refers to Sam Brook, the third edition of "Molecular Cloning Experiment Guide", Science Press, 2002; the basic process is shown in Figure 5) to The Arabidopsis non-coding protein gene AtIPS1 (database TAIR address: http://www.arabidopsis.org; gene accession number: At3g09922) was removed in order to construct a vector for transgenesis. To achieve the above goals, the following primers were designed:

IPS1OEF (forward primer):

5'-ACG GGTACC TGGCCATCCCCTAGCTAGGT-3' (the underlined sequence is the KpnI recognition site);

IPS1OER (reverse primer):

5'-TAC GGATCC CGGAAGCAAATTTACATGCACT-3' (the underlined sequence is BamHI recognition site);

MIM172F (forward primer):

5'-TT ATGCAGCATCGAGTTCAAAGATTCC AGCTTCGGTTCCCCTCGGAATCA-3' (the underlined sequence is the sequence encoding MIM172);

MIM172R (reverse primer):

5'-CT GGAATCTTGAACTCGATGCTGCAT AATTTCTAGAGGGAGATAAACA-3' (the underlined sequence is the complementary sequence encoding MIM172);

The total DNA of leaves of Col type Arabidopsis thaliana (from the Institute of Botany, Chinese Academy of Sciences) was extracted, and the DNA extraction method was the same as in Example 1. Using the DNA as a template, two sets of PCR were carried out in combination with primers IPS1OEF and MIM172R, MIM172F and IPS1OER respectively. The PCR system and program were consistent with those described in Example 1. Take 0.5 μl from each of the above two groups of PCR reaction products and mix them as the template for the next round of overlap extension PCR, and use primers IPS1OEF and IPS1OER as a combination for PCR amplification. The PCR system and program are the same as those described in Example 1. consistent with the procedure. Finally, a recombinant AtIPS1 fragment fused with MIM172 was obtained. The PCR product was detected by electrophoresis on 1% (mass/volume) TBE agarose gel, and the recovered PCR product was connected to the T-A cloning vector pGEMT-vector (purchased from Promega (Beijing) Biotechnology Co., Ltd. , Promega, USA), using the T7 and SP6 primers on the vector to carry out sequencing verification on the PCR product. The recombinant plasmid containing this PCR product was named plasmid TA-MIM172.

Embodiment 4: Construction of antagonist MIM172 overexpression vector

Use KpnI and BamHI to double-digest plasmid TA-MIM172 (the KpnI and BamHI were purchased from Bao Biological Engineering Dalian Co., Ltd., and the specific usage and dosage refer to the instructions of the corresponding products of the company. The plasmid TA-MIM172 is from Example 3, as shown in the figure 6), the 515bp DNA fragment containing MIM172 was recovered, and then combined with the vector plasmid pCAMBIA1301S [this vector has been publicly reported: Zhou et al., Over-expression of aspartate aminotransferase genes through KpnI and BamHI double digestion in rice resulted in altered nitrogen metabolism and increased amino acid content in seeds. Theor Appl Genet, 2009, 118:1381-1390; its basic skeleton was purchased from CAMBIA company in Australia (http://www.cambia.org/daisy/cambia /materials/overview.html) of pCAMBIA1301, by adding 35S promoter to realize the expression regulation of the transformed gene; its structure is shown in Figure B in Figure 6] using T4DNA ligase (purchased from Promega, specific usage and dosage refer to Instructions for the company's products) to connect. The ligation product was introduced into Escherichia coli DH10B (purchased from Promega Company) by electroporation (the electrotransformer is a product of eppendorf, and the voltage used in the present invention is 1800V, and the specific operation refers to the instruction manual of the instrument), and 400 μl LB medium was added for recovery 45min, spread on the LA medium plate containing 50mg/L kanamycin, incubate at 37°C for 14-16h (LA and LB formula reference: Sam Brook, "Molecular Cloning Experiment Guide" third edition, Science Press, 2002). Single clones were picked, expanded and cultured, and plasmids were extracted. Positive clones were screened by KpnI and BamHI double enzyme digestion, and the resulting expression vector was named 35S:MIM172, and its structure is shown in Figure 7.

Embodiment 5: the acquisition of transgenic rice

The expression vectors 35S:miR172bOE (from Example 2) and 35S:MIM172 (from Example 4) were introduced into the callus of rice variety Zhonghua 11 through the genetic transformation method mediated by Agrobacterium EHA105, after precultivation, infection, Co-cultivate, screen callus with resistance to hygromycin (an antibiotic used to screen positive transgenic callus, purchased from Roche Pharmaceutical Co., Ltd., Denmark), differentiate, take root and harden seedlings and transplant them in the field to obtain transgenic plants. The method of genetic transformation of Agrobacterium and the reagents and formulas used were optimized according to the report of Hiei et al. (Hiei et al., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T- DNA. Plant J, 1994, 6:271-282; Lin and Zhang, Optimizing the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell Rep, 2005, 23:540-548).

Agrobacterium-mediated genetic transformation reagent and formula involved in the present invention are as follows:

(1) Abbreviation of reagents and solutions

6-BA (6-BenzylaminoPurine, 6-benzyl adenine); KT (Kinetin, kinetin); NAA (Napthalene acetic acid, naphthalene acetic acid); IAA (Indole-3-acetic acid, indole acetic acid); 2, 4-D (2,4-Dichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid); AS (Acetosringone, acetosyringone); CH (Casein Enzymatic Hydrolysate, hydrolyzed casein); HN (Hygromycin B, hygromycin ); DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide); N6max (N6 large component solution); N6min (N6 small component solution); MSmax (MS large component solution); MSmin (MS small component solution)

(2) Main solution formula

1) N6max mother solution [10 times concentrated solution (10X)]

Dissolve one by one, then dilute to 1000ml at room temperature.

2) N6min mother liquor [100 times concentrated solution (100X)]

Dissolve at room temperature and dilute to 1000ml.

3) Fe ₂ -EDTA stock solution (100X)

Add 300ml of distilled water and 2.78g of ferric sulfate (FeSO ₄ 7H ₂ O) into a large triangular flask

Add 300ml of distilled water to another large triangular flask and heat it to 70°C, then add 3.73g of disodium ethylenediammonium tetraacetate (Na ₂ EDTA·2H ₂ O)

After they are all dissolved, mix them together, keep them in a water bath at 70°C for 2 hours, adjust the volume to 1000ml, and store them at 4°C for later use.

4) Vitamin storage solution (100X)

Add water to make up to 1000ml, and store at 4°C for later use.

5) MSmax stock solution (10X)

Dissolve at room temperature and dilute to 1000ml.

6) MSmin mother liquor (100X)

Dissolve at room temperature and dilute to 1000ml.

7) 2,4-D stock solution (1mg/ml)

2,4-D 100mg.

Dissolve 1ml of 1N potassium hydroxide for 5min, then add 10ml of distilled water to dissolve completely, then dilute to 100ml and store at room temperature.

8) 6-BA stock solution (1mg/ml)

6-BA 100mg.

Dissolve 1ml of 1N potassium hydroxide for 5min, then add 10ml of distilled water to dissolve completely, then dilute to 100ml and store at room temperature.

9) NAA stock solution (1mg/ml)

NAA 100mg.

Dissolve 1ml of 1N potassium hydroxide for 5 minutes, then add 10ml of distilled water to dissolve completely, then dilute to 100ml, and store at 4°C for later use.

10) IAA stock solution (1mg/ml)

IAA 100mg.

Dissolve 1ml of 1N potassium hydroxide for 5 minutes, then add 10ml of distilled water to dissolve completely, then dilute to 100ml, and store at 4°C for later use.

11) Glucose stock solution (0.5g/ml)

Glucose 125g

Dissolve in distilled water to a volume of 250ml, and store at 4°C after sterilization.

12) AS stock solution

AS 0.392g

DMSO 10ml

Aliquot into 1.5ml centrifuge tubes and store at 4°C for later use.

13) 1N potassium hydroxide stock solution

Potassium hydroxide (KOH) 5.6g

Dissolve in distilled water and make up to 100ml, store at room temperature for later use.

14) KT stock solution (1mg/ml)

KT 100mg.

Dissolve 1ml of 1N potassium hydroxide for 5min, then add 10ml of distilled water to dissolve completely, then dilute to 100ml and store at room temperature.

(3) Medium formula

1) Induction medium

Add distilled water to 900ml, adjust the pH value to 5.9 with 1N potassium hydroxide, boil and set the volume to 1000ml, dispense into 50ml Erlenmeyer flasks (25ml/bottle), seal and sterilize.

2) subculture medium

Add distilled water to 900ml, adjust the pH value to 5.9 with 1N potassium hydroxide, boil and set the volume to 1000ml, dispense into 50ml Erlenmeyer flasks (25ml/bottle), seal and sterilize.

3) Pre-medium

Add distilled water to 250ml, adjust the pH value to 5.6 with 1N potassium hydroxide, seal and sterilize.

Heat to dissolve the culture medium before use, add 5ml of glucose stock solution and 250μl of AS stock solution, and pour them into petri dishes (25ml/dish).

4) Co-culture medium

Add distilled water to 250ml, adjust the pH value to 5.6 with 1N potassium hydroxide, seal and sterilize.

Heat to dissolve the culture medium before use, add 5ml of glucose stock solution and 250μl of AS stock solution, and pour them into petri dishes (25ml/dish).

5) Suspension medium

Add distilled water to 100ml, adjust the pH value to 5.4, divide into two 100ml Erlenmeyer flasks, seal and sterilize.

Add 1 ml of glucose stock solution and 100 μl of AS stock solution before use.

6) Select medium

Add distilled water to 250ml, adjust the pH value to 6.0, seal and sterilize.

Dissolve the culture medium before use, add 250 μl of 50 mg/ml hygromycin and 400 ppm cephalosporin, and pour them into petri dishes (25 ml/dish).

7) Pre-differentiation medium

Add distilled water to 250ml, adjust the pH value to 5.9 with 1N potassium hydroxide, seal and sterilize.

Dissolve the culture medium before use, add 250 μl of 50 mg/ml hygromycin and 400 ppm cephalosporin, and pour them into petri dishes (25 ml/dish).

8) Differentiation medium

Add distilled water to 900ml, and adjust the pH value to 6.0 with 1N potassium hydroxide.

Boil and set the volume to 1000ml, dispense into 100ml Erlenmeyer flasks (50ml/bottle), seal and sterilize.

9) Rooting medium

Add distilled water to 900ml, and adjust the pH value to 5.8 with 1N potassium hydroxide.

Boil and set the volume to 1000ml, dispense into rooting tubes (25ml/tube), seal and sterilize.

10) LA medium (LB medium does not contain agar powder)

Dissolve in distilled water to a volume of 250ml, put in a 500ml Erlenmeyer flask, and store at room temperature after sterilization for later use.

The steps of the Agrobacterium-mediated genetic transformation involved in the present invention are as follows:

(1) Callus induction

1) Ripe rice seeds are dehulled, then treated with 70% ethanol for 1 min and 0.15% mercury chloride (HgCl ₂ ) for 15 min

2) Wash the seeds with sterilized water 4-5 times;

3) seeds are placed on the induction medium;

4) Culture in a dark place for 5 weeks at a temperature of 26±1°C.

(2) Callus subculture

Select bright yellow, compact and relatively dry embryogenic calli, and place them on the subculture medium for 2 weeks in the dark at a temperature of 26±1°C.

(3) Pre-cultivation

Select compact and relatively dry embryogenic calli, place them on the pre-medium and culture them in the dark for 4 days at a temperature of 26±1°C.

(4) Agrobacterium culture

1) Pre-cultivate Agrobacterium EHA1052d containing the constructed vector on LA medium with kanamycin at a temperature of 28°C;

2) Transfer the Agrobacterium to the suspension medium, and culture on a shaker at 28° C. for 2-3 hours.

(5) Agrobacterium infection

1) transfer the pre-cultured callus to a sterilized bottle;

2) adjust the suspension of Agrobacterium to OD ₆₀₀ 0.8-1.0;

3) Soak the callus in the Agrobacterium suspension for 30 minutes;

4) Transfer the callus to a sterilized filter paper and blot dry; then place it on a co-culture medium for 2 days at a temperature of 19-20°C.

(6) Callus washing and selection culture

1) Wash the callus with sterilized water until the Agrobacterium cannot be seen;

2) Soak in sterilized water containing 400ppm cephalosporin for 30min;

3) transfer the callus to the sterilized filter paper and blot dry;

4) Transfer the callus to the selection medium for selection 2-3 times, each time for 2 weeks. (The first cephalosporin screening concentration is 400ppm, and the second and later is 250ppm)

(7) differentiation

1) Transfer the resistant callus to the dark place on the pre-differentiation medium and culture it for 5-7d;

2) Transfer the pre-differentiation cultured callus to the differentiation medium, and culture it under light at 26° C. for 5-7 weeks.

(8) Rooting

1) pull out the well-differentiated seedlings, and cut off the roots produced during differentiation;

2) Then transfer it to the rooting medium and cultivate it under light for 2-3 weeks at a temperature of 26°C.

(9) Transplanting

Wash off the residual medium on the roots and transfer the seedlings with a good root system to the greenhouse while keeping them moist for the first few days. After hardening the seedlings in the greenhouse for about 2 weeks, they are then transferred to the field.

Example 6: Identification and phenotype observation of transgenic rice

The total DNA of the leaves of the T0 generation 35S:miR172bOE and 35S:MIM172 transformed plants (from Example 5) was extracted, and then the positive detection of the T0 generation transformed plants was performed by PCR method. The method system of DNA extraction and PCR is the same as that in Example 1.

Utilize β-glucuronidase gene (GUS) primer to carry out positive detection, the sequence of GUS primer is as follows:

GUS-F (forward primer): 5'-CCAGGCAGTTTTAACGATCAGTTCGC-3'

GUS-R (reverse primer): 5'-GAGTGAAGATCCCTTTCCTTGTTACC-3'

The results of PCR detection showed that the GUS-positive plants from the expression vector 35S:miR172bOE transgene, compared with the negative plants, all showed reduced panicle branches, decreased panicle number, decreased leaf width, and decreased leaf length. The morphological changes are shown in Figure 8 and Figure 10; while the GUS-positive plants transgenic to the expression vector 35S:MIM172 all showed shorter plant height, the number of primary branches per ear, and the number of secondary branches per ear. , number of spikelets per panicle, leaf length and leaf width all increased significantly, and the phenotypic changes are shown in Figure 9 and Figure 10.

The positive plants of the T0 generation were divided into individual plants to collect seeds, and the transgenic materials of the T1 generation were continued to be planted. The plants of the T1 generation were also subjected to PCR amplification of the GUS gene to detect the negative and positive of the isolated individual plants, and the transgenic events and phenotypic changes were monitored. Co-segregation detection, and statistical phenotypic data.

In order to perform phenotypic analysis, the applicant sowed the seeds of T0 plants (from Example 5) that were independently transformed from each of the expression vectors 35S:miR172bOE and 35S:MIM172 in the seedling field after soaking and accelerating germination, and transplanted to the seedling field after 20 days. Daejeon obtained T1 transgenic plants. The planting density is 15cm×24cm, and the planting site is the experimental field of Huazhong Agricultural University in Hongshan District, Wuhan City, Hubei Province, China. The field management is carried out according to the conventional rice planting method under the condition of a safety protection facility. The PCR method was used to detect the GUS gene for negative and positive identification, and the corresponding relationship between the PCR test results and the phenotype was analyzed. The results showed that in the T1 generation, all the GUS-positive individual plants isolated from the transgenic offspring of the carrier 35S:miR172bOE showed reduced panicle branches, decreased panicle number, decreased leaf width, and decreased leaf length compared with the negative individual plants. It was suggested that transgenic events for miR172b co-segregate with these phenotypic changes. However, all the GUS-positive individual plants isolated from the transgenic progeny of the carrier 35S:MIM172 showed shorter plant height, the number of primary branches per panicle, the number of secondary branches per panicle, and the number of spikelets per panicle. , leaf length and leaf width were significantly increased, indicating that the transgenic events of MIM172 were also co-segregated with these phenotypic changes.

Investigate and record the phenotypic changes and data of T1 generation related plants, including plant height, number of primary branches per panicle, number of secondary branches per panicle, number of spikelets per panicle, length and width of flag leaves, and separate them for each family The GUS-negative individual plants were used as controls, and statistical analysis was performed respectively.

Table 1 Phenotype statistics of positive and negative individual plants of 35S:miR172bOE (miR172OE) and 35S:MIM172 (MIM172) transgenic T1 generation.

Explanation of Table 1: (+) and (-) represent transgene positive and negative individual plants, respectively. Statistical analysis was carried out by t test, ^{a, b, c} represent the significant levels of P<0.05, 0.01 and 0.001, respectively.

Example 7: Analysis of the expression level of miR172 in transgenic rice

In order to analyze the impact of 35S:miR172bOE and 35S:MIM172 transgenic events on the expression of endogenous miR172, the applicant further used the method of stem-loop RT-PCR (Shen et al., Global expression profiling of rice microRNAs by one-tube stem- loopreverse transcription quantitative PCR revealed important roles of microRNAs in abiotic stress responses. MolGenet Genomics, 2010, 284:477-488) detected the expression level of miR172 in transgenic plants. Its specific operation method is as follows:

(1) Extract the RNA from the tillering stage leaves of the transformed plants of 35S:miR172bOE and 35S:MIM172, the reagent used for RNA extraction is the Trizol extraction kit purchased from Invitrogen Company (see the kit instruction manual for specific operation steps);

(2) Reverse transcription to synthesize the first strand of cDNA, the steps are as follows:

1) Take 200ng of extracted total RNA, add DNaseI 1μl, 10xDNaseI buffer 1μl, add DEPC (diethylpyrocarbonate, strong inhibitor of RNase, working concentration is 0.01%) treated water to 10μl, mix well Place at room temperature for 15 minutes to remove residual genomic DNA; 2) Add 1 μl of 0.2M EDTA after 15 minutes, and incubate in a water bath at 65°C for 10 minutes to remove DNaseI activity; 3) Add 1 μl of primer miR172stemloopR (concentration: 10 mM, sequence: 5 '-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATGCAG-3'), or 1 μl primer U6R (concentration: 10mM, sequence: 5'-GGACCATTTCTCGATTTGTACGTG-3'), and incubated in a water bath at 65°C for 10min to destroy the secondary structure of RNA, and then placed on ice for 5min ; 4) Add 4 μl of 5x first strand buffer, 2 μl of 0.1M DTT (mercaptoethanol), 1 μl of 10mM dNTP mixture, and 1 μl of reverse transcriptase, mix well and place in a water bath at 42°C for 1.5 hours; The reverse transcription product was placed in a dry bath at 90°C for 3 minutes to inactivate the reverse transcriptase; 6) 120 μl of water was added to the reverse transcription product, and the final reaction product was stored at -20°C after mixing. All reagents used in the reaction were purchased from Invitrogen.

(3) Real-time fluorescent quantitative PCR, the steps are as follows:

The expression of miR172 was detected by real-time fluorescent quantitative PCR on the obtained reverse transcription product (the primer combination used was 172QRTF and miRUniverseR), and the expression level of U6 (the primer combination used was U6F and U6R) was used as the internal reference for balance change. Reagents related to real-time fluorescence quantitative PCR were purchased from Bao Bioengineering Dalian Co., Ltd., and the reaction system was referred to the instruction manual. The PCR machine was 7500 from ABI Company in the United States. The PCR parameters were pre-denaturation at 95°C for 10s, denaturation at 95°C for 5s after entering the cycle, annealing and extension at 60°C for 40s, and 45 cycles. The primer sequences used in real-time fluorescent quantitative PCR are:

172QRTF (forward primer): 5'-GGCGCAGAATCTTGATGATG-3'

miRUniverseR (reverse primer): 5'-CCAGTGCAGGGTCCGAGGT-3'

U6F (forward primer): 5'-TACAGATAAGATTAGCATGGCCCC-3'

U6R (reverse primer): 5'-GGACCATTTCTCGATTTGTACGTG-3'

miR172 in rice is encoded by 4 precursor genes, in view of the high identity of the miR172 mature body sequences formed by these 4 precursor genes (respectively miR172a, miR172b, miR172c and miR172d, there are at most 2 base difference), the above real-time fluorescent quantitative PCR results cannot distinguish these different miR172 members, and the detection is the overall result of all miR172 in the body. The results of real-time fluorescent quantitative PCR showed that in the 35S:miR172bOE transgenic plants, miR172 was up-regulated, and the results were shown in Figure 11; while in the 35S:MIM172 transgenic plants, miR172 was down-regulated, and the results were shown in Figure 12.

Example 8: Detection of miR172 antagonism effect by MIM172

In order to further analyze whether MIM172 can antagonize the effect of miR172, the applicant crossed 35S:miR172bOE and 35S:MIM172 transgenic plants with each other, and analyzed the hybrid relative to the wild type, 35S:miR172bOE transgenic plants and 35S:MIM172 transgenic plants with panicle type as a representative The phenotypic changes of the plants showed that the panicle type of the hybrid was similar to that of the 35S:MIM172 transgenic plant, and the number of panicle branches and spikelets increased compared with the wild type, so the number of panicle branches and spikelets decreased due to the 35S:miR172bOE transgene The isophenotype was restored by the 35S:MIM172 transgene, that is, MIM172 can antagonize the effect of miR172, and the results are shown in FIG. 13 .

Claims (7)

1. a kind of application of gene of microRNA172b in the plant height of adjusting and controlling rice, fringe type and blade dimensions, it is characterised in that the core of the gene Nucleotide sequence such as SEQ ID NO:Shown in 1.

2. a kind of genetic fragment of the precursor-gene pri-microRNA172b of coding microRNA172b is in the plant height of adjusting and controlling rice, fringe type and blade chi Application in very little, it is characterised in that the fragment is SEQ ID NO:Sequence described in 2.

3. a kind of genetic fragment of the antagonist MIM172 of microRNA172, it is characterised in that the nucleotide sequence of the genetic fragment such as SEQ ID NO:Shown in 3.

4. the microRNA172 antagonist MIM172 genetic fragments described in claim 3 are in the application in rice modification.

5. the application described in claim 4, including the application in the plant height of adjusting and controlling rice, fringe type and blade dimensions.

6. one kind includes SEQ ID NO:The plant expression vector of sequence shown in 2.

7. one kind includes SEQ ID NO:The plant expression vector of sequence shown in 3.