CN119286911A

CN119286911A - A Halophilous Iris IhCHS1 gene for improving plant salt tolerance and jasmonic acid accumulation ability and its application

Info

Publication number: CN119286911A
Application number: CN202411521239.3A
Authority: CN
Inventors: 刘清泉; 原海燕; 顾希; 张永侠; 王银杰; 张婷; 刘璇
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2024-10-29
Filing date: 2024-10-29
Publication date: 2025-01-10
Anticipated expiration: 2044-10-29
Also published as: CN119286911B

Abstract

The present invention relates to a salt-loving iris IhCHS1 gene and its application for improving the salt tolerance and jasmonic acid accumulation ability of plants. The salt-loving iris IhCHS1 gene belongs to the plant chalcone synthase (CHS) family. The present invention constructs an overexpression vector of the IhCHS1 gene and transfers it into salt-sensitive plants. Through growth phenotype, physiological analysis and metabolite analysis, it is found that under salt stress, compared with the wild type, the root length, mature biomass and flower stem height of the plant seedlings overexpressing the IhCHS1 gene are significantly increased, and the content of jasmonic acid compounds is significantly increased, indicating that overexpressing the IhCHS1 gene can significantly enhance the salt tolerance and jasmonic acid accumulation of plants, and can be used to improve the growth ability of plants under salt stress and the synthesis ability of jasmonic acid compounds. The salt-loving iris IhCHS1 gene and its overexpression vector have important application value in the field of plant salt resistance and plant secondary metabolism gene engineering, and can be directly applied to the creation of new salt-tolerant germplasms of the genus Iris and even other plants.

Description

Salt-loving iris IhCHS gene for improving accumulation capacity of plant salt tolerance and jasmonate and application

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a salt-like iris IhCHS gene for improving the accumulation capacity of salt tolerance and jasmonate substances of plants and application thereof.

Background

The saline-alkali soil is an important land resource in China, and the restoration improvement and the development and the utilization of the saline-alkali soil have important significance for ecological environment construction and agricultural sustainable development. By developing the key genes related to the high salt stress tolerance of halophytes, the action mechanism of the halophytes is clarified, the halophytes are transferred into salt sensitive plants, new germplasm of the halophytes is cultivated, new germplasm and new varieties of the high salt tolerant plants are cultivated, and ecological restoration and resource development of saline-alkali soil can be effectively promoted.

Research shows that iris plants have certain stress resistance to saline-alkali soil and are good materials for ecological restoration of saline-alkali soil, but different varieties of plants can form different ecological systems, and cultivation of different varieties of salt-tolerant plants is helpful for recovering and protecting the biodiversity of the saline-alkali soil, and the diversity is helpful for improving the stability and adaptability of the whole ecological system, so how to cultivate the salt-tolerant plants of different varieties is important.

Chalcone synthase (EC 2.3.1.74, chs) is the first rate-limiting enzyme and key enzyme of the flavonoid biosynthetic pathway and belongs to the family of plant type III polyketide synthases. The CHS gene was first isolated from parsley and subsequently cloned in a variety of monocotyledonous, dicotyledonous and gymnosperm plants. CHS has various physiological functions in plants due to the participation of biosynthesis of secondary metabolites such as flavonoids, such as participation in plant flower color formation, regulation of plant pollen fertility, and the like. However, the function and mechanism of action of the iris tectorum CHS gene (IhCHS 1) in regulating plant salt tolerance and jasmonic acid accumulation are not clear at present, and the functional study of CHS gene in regulating plant jasmonic acid synthesis is not reported in the related literature.

Disclosure of Invention

In order to solve the technical problems, the IhCHS gene is obtained through excavation and screening, and compared with other genes, the salt tolerance and jasmonate accumulation capacity of plants can be remarkably improved through over-expression of the gene, and the improvement of the growth capacity of the plants under salt stress tolerance and the synthesis capacity of one or more substances selected from jasmonic acid, jasmonic acid derivatives and precursor substances 12-oxo-plant dienoic acid is mainly reflected. The transformation method provided by the invention provides a brand-new mode for improving the salt stress tolerance and the jasmonic acid biosynthesis capability of plants.

A first object of the present invention is to provide a method for improving salt tolerance and/or jasmonate accumulation in plants, comprising the steps of:

Overexpressing a chalcone synthase encoding gene in the plant (IhCHS 1);

The amino acid sequence of the chalcone synthase is shown as SEQ ID NO. 1.

Further, the plants include Iridaceae and Brassicaceae, preferably the Iridaceae includes Iridaceae, the Brassicaceae includes Arabidopsis, and most preferably the plant is Arabidopsis.

Further, the sequence of the chalcone synthase coding gene is shown as SEQ ID NO. 2.

The second object of the present invention is to provide the use of a nucleic acid molecule encoding a chalcone synthase having an amino acid sequence as shown in SEQ ID NO.1 for the preparation of a product for improving salt tolerance and/or jasmonate accumulation in plants.

Further, the improvement of the plant salt tolerance and/or jasmonate accumulation capacity comprises improvement of the plant growth capacity under salt stress tolerance and improvement of the synthesis capacity of one or more substances selected from the group consisting of plant jasmonic acid, jasmonic acid derivatives and precursor substances 12-oxo-plant dienoic acid. Salt stress is one of the most serious abiotic stresses in nature, and plants gradually form a series of adaptive mechanisms in the process of suffering from salt stress, such as regulating the absorption and transportation of salt ions through hormone signal transduction pathways, maintaining the balance of the salt ions in cytoplasm, inducing an antioxidant protection system, accumulating osmotic regulating substances, reducing cell osmotic potential and the like. The jasmonate is a fatty acid derivative containing cyclopentanone basic structure, and mainly comprises free jasmonate and derivative thereof, active precursor substance 12-oxygen-plant dienoic acid and other compounds. Jasmonates are important growth regulating substances and hormone signal molecules in plants, and have very important roles in regulating the growth and development of plants, promoting the flower bud differentiation and flowering process, resisting abiotic stress, resisting disease reaction and the like. In addition, jasmonates have many applications in perfumery and cosmetics.

Further, the product contains a DNA sequence or an RNA sequence (such as mRNA) for encoding the chalcone synthase, and the DNA sequence for encoding the chalcone synthase is shown as SEQ ID NO. 2.

The invention also provides a product for cultivating salt-tolerant and/or high jasmonic acid accumulation type plants, which contains a nucleic acid molecule for encoding chalcone synthase, wherein the amino acid sequence of the chalcone synthase is shown as SEQ ID NO. 1.

A third object of the present invention is to provide a method for breeding a transgenic plant comprising the steps of expressing a chalcone synthase encoding gene in a recipient plant to obtain the transgenic plant;

The transgenic plant comprises a salt tolerant and/or highly jasmonate accumulating plant or plant model;

The amino acid sequence of the chalcone synthase is shown as SEQ ID NO. 1.

Further, the method specifically comprises the steps of introducing a chalcone synthase encoding gene into a recipient plant, and culturing until a homozygote expressing the chalcone synthase encoding gene is obtained, thereby obtaining the transgenic plant.

The fourth object of the invention is to provide a chalcone synthase for improving salt tolerance and/or jasmonate accumulation capability of plants, wherein the amino acid sequence of the chalcone synthase is shown as SEQ ID NO. 1.

Further, the salt tolerance and/or jasmonate accumulation capacity includes the plant's ability to grow under salt stress tolerance and the ability to synthesize one or more of jasmonic acid, jasmonic acid derivatives and precursor substance 12-oxo-plant dienoic acid.

It is a fifth object of the present invention to provide a nucleic acid molecule encoding said chalcone synthase.

Further, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 2.

It is a sixth object of the present invention to provide a recombinant expression vector containing said nucleic acid molecule.

Further, the backbone of the expression vector includes, but is not limited to, pBI121, and the promoter includes, but is not limited to, 35S.

It is a seventh object of the present invention to provide recombinant cells containing said nucleic acid molecules.

Further, host cells include, but are not limited to, plant cells.

By means of the scheme, the invention has at least the following advantages:

The invention takes halophyte Iris philippinensis as a material, and clones IhCHS genes related to salt response of the Iris philippinensis by a PCR technology. Further adopting homologous recombination technology to construct a plant over-expression vector pBI121-IhCHS1, and realizing the over-expression of IhCHS gene in salt-sensitive plant Arabidopsis thaliana by a method of infecting inflorescence with agrobacterium. Under salt stress, the root length, fresh weight, dry weight, flower stem height and jasmonic acid compound content of the arabidopsis seedlings over-expressed IhCHS1 genes are obviously increased compared with that of wild arabidopsis, which shows that the IhCHS1 genes can obviously enhance the salt tolerance and jasmonic acid accumulation of transgenic arabidopsis, can be used for improving the growth and jasmonic acid biosynthesis capacity of plants under salt stress, and has important application value in the fields of plant salt resistance and plant secondary metabolism genetic engineering.

The foregoing description is only an overview of the present invention, and is presented in terms of preferred embodiments of the present invention and the following detailed description of the invention in conjunction with the accompanying drawings.

Drawings

In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings.

FIG. 1 shows fold changes in expression of different CHS genes of Iris salina after salt stress treatment;

FIG. 2 is a schematic diagram of the secondary structure of the protein IhCHS of Iris salina;

FIG. 3 is a schematic diagram of the tertiary structure of iris tectorum schizandrum IhCHS protein;

FIG. 4 shows construction of a transgenic plant PCR identification of iris salina IhCHS gene overexpression vector;

FIG. 5 is the effect of salt stress on root elongation of wild type and transgenic IhCHS Arabidopsis seedlings;

FIG. 6 is an effect of salt stress on growth of a wild type and transgenic IhCHS A.thaliana mature period flower stem;

FIG. 7 is the effect of salt stress on wild-type and transgenic IhCHS A.thaliana maturity biomass;

FIG. 8 is the effect of over-expression IhCHS of the 1 gene on accumulation of transgenic Arabidopsis jasmonates.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

The scheme of the invention is as follows:

(1) Screening of key genes according to the invention, screening of the salt-loving iris gene expression profile (obtained by transcriptome sequencing) under salt stress shows that IhCHS gene expression is significantly up-regulated under salt stress compared with other genes (figure 1), so that IhCHS gene is over-expressed in subsequent selection, and transgenic experiments are carried out to improve salt tolerance of plants.

(2) Constructing a plant over-expression vector of IhCHS gene;

(3) Transforming the constructed plant over-expression vector (pBI 121-IhCHS 1) into a plant body;

(4) Positive transgenic plants are screened by utilizing a molecular biological technology, salt-tolerant plants are indeed obtained through physiological and metabonomic technology identification, and the jasmonate accumulation capacity of the plants can be remarkably enhanced.

The sequence information related in the embodiment of the invention is as follows:

IhCHS1 protein sequence (SEQ ID NO. 1)

MVNVAELCKLQRAEGPAAILAIGTANPPNAVSQSEYPDYYFRITNSEHKHELKAKFKRMCDNSMIKKRYMYLTEEILKENPSLCAYMAPSLDVRQDMVVVEVPKLGKEAAAKAIKEWGQPKSKITHVVFCTTSGVDMPGADYQLTRLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAENNRGARVLVVCSEITAVTFRGPSDSHLDSLVGQALFGDGASALIVGADPIENVERPLFEIVSAAQTILPDSEGAIDGHVREAGLTFHLLKDVPGIISKNIEKSLEEAFKPLGIADWNSLFWVAHPGGPAILDQVEAKLGLKPEKLRATRHVLSEYGNMSSACVLFILDEMRKRSVEAGNGTTGEGLEWGVLFGFGPVAAA

IhCHS1 Gene sequence (SEQ ID NO. 2)

atggtgaatgttgcagaactatgcaagctgcagagggctgagggcccggccgccatcttggccatcggcacggccaatccgcccaacgcagtgtcgcagagcgagtatccagactactacttcaggatcaccaacagcgagcacaaacatgagctcaaagccaagttcaagcgcatgtgcgacaactcgatgatcaagaagaggtacatgtacttgactgaggagatcctgaaagaaaaccctagcctttgcgcctacatggccccttccctcgacgtgcgtcaagacatggtcgtcgtcgaggtccccaagctcggcaaagaggccgccgccaaggccatcaaggagtgggggcagcccaagtccaagatcacccacgtcgtcttctgcaccacaagcggcgtcgacatgcccggggccgactaccagctcacccgcctcctcggccttcgcccctccgtcaagcgcctcatgatgtaccagcagggatgcttcgccggcggcacggtgctccgcctcgccaaggacctcgccgagaacaaccgcggcgcgcgtgttcttgtcgtgtgttccgagatcaccgctgtcaccttccgcggaccttccgactcccacctcgacagcctggtaggccaggccctgttcggagacggtgcttctgctctcattgtaggcgccgaccctatcgagaacgtcgagcggccgctcttcgagatcgtctccgctgcacaaacgatactgcccgacagcgagggggcgatcgatggccacgtaagggaagcgggcctgacgttccacctgctcaaggatgtccccgggatcatatcgaagaacatcgagaagagcctcgaggaggcgttcaagccgctagggatcgcggattggaactcattgttctgggtcgcccacccgggaggaccggctatactggaccaggtcgaggccaagctcgggctgaagccggagaagctgagggccacaaggcacgtgctgagcgagtacgggaacatgtccagtgcttgcgtgctcttcatcctcgacgagatgaggaagcggtcggtcgaggccgggaatgggaccaccggtgaggggctcgagtggggggtgctcttcgggttcggtccggttgctgctgcgtga

EXAMPLE 1 physical and chemical Properties and Structure analysis of Iris philippica IhCHS1

1. Based on the amino acid sequence encoded by IhCHS gene, the Molecular Weight (MW), theoretical isoelectric point (pI) and extinction coefficient of IhCHS protein were predicted by using pI/MW (http:// web. Expasy. Org/computer_pi /) and Peptide and Protein Molecular Weight Calculator(https://www.aatbio.com/tools/calculate-peptide-and-protein-molecular-weight-mw) online program, respectively, and subcellular localization of IhCHS protein was predicted by Cell-PLoc 2.0.0 (http:// www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2 /) online tool. The results show that the iris tectorum-hollandii chalcone synthase IhCHS protein contains 379 amino acids, the predicted Molecular Weight (MW) of IhCHS protein is 41.228kDa, the isoelectric point (pI) is 6.28, the extinction coefficient is 35120M ^-1cm^-1, and the predicted subcellular localization is in cytoplasm.

Further utilize SignalP

(Https:// services. Healthcare. Dtu. Dk/services/SignalP-6.0 /) the signal peptide was predicted for the amino acid sequence of Iris philippica IhCHS. The results showed that the signal peptide probability was low, indicating that the amino acid sequence of Iris philica IhCHS may not contain signal peptide.

2. The secondary and tertiary structures of the IhCHS protein were predicted using online analysis tools PSIPRED (http:// bioif. Cs. Ucl. Ac. Uk/psipred /) and SWISS-MODEL (https:// swissmodel. Expasy/interactive), respectively.

As shown in FIG. 2, PSIPRED predicted results show that the secondary structure of the IhCHS protein contains 19 alpha helices and 8 beta sheets, and contains the characteristic sequence of chalcone synthase (RLMMYQQGCFAGGTVLR) and four conserved catalytic active sites (Cys 164, phe 215, his 303 and Asn 336), and that in addition, a typical chalcone synthase receptor binding pocket is present in the 3D structure of the IhCHS protein (FIG. 3). The structural analysis above shows that IhCHS protein may have the catalytic activity of chalcone synthase.

EXAMPLE 2 cloning of the Iris salicifolia IhCHS Gene and construction of plant overexpression vector

1. Seeds of Iris salicifolia (Iris halophila pall.) were sterilized by immersing in 5% (v/v) sodium hypochlorite and then washed 5 times with ddH ₂ O. After sterilization, the seeds were planted in a container containing clean moist sand for germination. When the seedlings reached a height of about 10cm, they were transferred to a plastic cup containing 1/2Hoagland nutrient solution for cultivation, and the incubator was illuminated at an intensity of 22,500Lux. Seed germination and seedling growth conditions were 25/22 ℃ (day/night temperature), 60-80% (relative humidity) and 14/10-h (day/night light cycle). After one week of culture, the aerial parts and root systems of the Iris philippinensis seedlings are respectively collected after being treated by a nutrient solution containing 300mM NaCl for 24 hours, and the aerial parts and root systems are frozen by liquid nitrogen and used for extracting total RNA.

2. Total plant RNA extraction kit (TaKaRa, china) was used to extract Iris salina seedling RNA. The procedure was followed according to the kit instructions, using RNase-free DNase I (TaKaRa, china) to remove genomic DNA during the extraction. After the extracted RNA is qualified, cDNA is synthesized by using a reverse transcription kit (TaKaRa, china).

3. The mRNA sequence obtained by sequencing the full-length transcriptome is respectively designed into an upstream primer and a downstream primer for amplifying the full-length CDS of IhCHS genes, the XbaI sequence on a plant over-expression vector pBI121 and a sequence (5'-gagaacacgggggactctagaATGGTGAATGTTGCAGAACTATGC-3') with the length of 15bp upstream of the locus thereof are added at the 5 'end of the upstream primer, the SmaI sequence and a sequence (5'-ataagggactgaccacccgggTCACGCAGCAGCAACCGG-3') with the length of 15bp downstream of the locus thereof are added at the 5' end of the downstream primer, and the wild iris cDNA is used as a template to clone the IhCHS genes by PCR. And (3) detecting the PCR product by 1% agarose gel electrophoresis, and then performing rubber tapping recovery. At the same time, the pBI121 plasmid was digested with XbaI and SmaI, and the digested products were recovered. Utilizing homologous recombinase ExnaseTMII%IIOne Step Cloning Kit, vazyme) and the PCR recovery product are connected with the digestion recovery product, the connection product is transformed into escherichia coli competent DH5a, and sequencing results show that the IhCHS gene fragment is successfully connected with the digestion site of pBI121, which shows that the plant over-expression vector pBI121-IhCHS1 is successfully constructed (figure 4A), and the sequence of the target fragment is determined to be SEQ ID NO.2.

Example 3 genetic transformation of Iris salicifolia IhCHS1 and functional analysis thereof in salt tolerance in Arabidopsis thaliana

1. Agrobacterium GV3101 containing recombinant plasmid pBI121-IhCHS1 stored in-80 ℃ refrigerator is inoculated in YEB solid culture medium (containing Kan:50 μg/mL; rif:50 μg/mL), dark cultured for 48h at 28 ℃, the activated Agrobacterium is inoculated in 5mL YEB liquid culture medium (containing Kan:50 μg/mL; rif:50 μg/mL), shake cultured for 24h at 28 ℃, 2mL of the above bacterial liquid is absorbed in 200mL YEB liquid culture medium (containing Kan:50 μg/mL; rif:50 μg/mL), shake cultured at 28 ℃ at 250rpm, when OD600 reaches 1.0-1.2, bacterial liquid is sub-packaged in 50mL, 4 ℃ and 4000rpm are centrifuged for 10min, 5% sucrose is added for resuspension, and centrifugation is carried out at 4000rpm for 10min again, and then the supernatant is obtained after sedimentation, thus obtaining the bacterial body. Adding 5% sucrose solution containing 0.02% surfactant (silwet L-77) to make the OD600 value of the bacterial solution reach about 0.8-1.0, soaking the arabidopsis inflorescence in the agrobacterium bacterial solution for about 1-5min, taking out the arabidopsis, sucking the bacterial solution on the stems with absorbent paper, wrapping with preservative film to increase humidity, culturing in darkness for 24h, removing the preservative film, performing conventional culture, spraying water for 1-2 times per day, and increasing humidity. And (4) harvesting first generation transgenic IhCHS gene seeds when the pods of the arabidopsis thaliana to be transformed are yellow, drying at 37 ℃, and storing in a 4 ℃ refrigerator.

2. Sowing Arabidopsis seeds on MS solid culture medium containing Kan (50 mug/ml), placing in an incubator for culturing, transferring seedlings which can normally grow on a screening culture medium into a nutrient medium for culturing and breeding, cutting transgenic Arabidopsis seedling leaves after four weeks, extracting genome DNA, taking Arabidopsis DNA as a template, carrying out PCR identification by using specific primers on a carrier and a target gene, and using plants which are positive through PCR identification for subsequent homozygote screening and transcription level detection. And finally screening T3 generation single copy insertion homozygotes IhCHS-OE 1 and IhCHS-OE 2 for subsequent experiments by counting kanamycin resistance character segregation ratio of PCR positive offspring plants.

3. In order to judge whether the transcription of IhCHS gene in transgenic Arabidopsis IhCHS-OE 1 and IhCHS1-OE2 can be normally performed or not, RNA of wild type and transgenic Arabidopsis is respectively extracted, two specific primers are designed, and the transcription level is detected by using a fluorescent quantitative PCR method.

The results showed that in wild type Arabidopsis, the IhCHS gene was not detected, whereas the IhCHS gene was highly expressed in both transgenic plants IhCHS-OE 1 and IhCHS-OE 2, and the above study results showed that the Iris salina IhCHS1 gene had successfully achieved overexpression in Arabidopsis (FIG. 4B), and the next biological function analysis was possible.

4. The T3 generation homozygote of the transgenic IhCHS gene and the wild arabidopsis seeds are respectively sown in an MS culture medium (containing 1% of sucrose and 1% of agar) after being disinfected, and are placed in an illumination incubator for vertical culture after 3d synchronization at 4 degrees, wherein the culture conditions are that the temperature of day/night is 22 ℃ and 20 ℃, the illumination is provided by an LED lamp, the period is 16/8 hours (day/night), and the relative humidity of air is 60-80% (RH). After growing for 2 days, selecting wild type and transgenic IhCHS1 Arabidopsis plants with consistent growth, respectively transplanting the wild type and transgenic 3835.1 Arabidopsis plants to a culture medium containing 100mM, 150mM and 175mM NaCl, taking a normal culture medium as a control, and taking root length after growing for 2 weeks, so as to test the influence of over-expression of the Iris salicifolia chalcone synthase gene IhCHS on the root elongation of Arabidopsis seedlings.

As shown in FIG. 5, there was no significant difference between the growth of transgenic IhCHS A.thaliana and that of wild type A.thaliana under normal culture and 100mM NaCl treatment conditions, and no significant difference between the root lengths, whereas under high salt (150 mM and 175mM NaCl) treatment conditions, transgenic IhCHS A.thaliana and wild type growth were significantly inhibited, but the inhibition levels were significantly different between them, and transgenic IhCHS A.thaliana root length was significantly greater than that of wild type, wherein after 2 weeks of 150mM NaCl treatment, the root elongations of IhCHS-OE 1 and IhCHS1-OE2 transgenic lines were 86.66% and 114.97% higher, respectively, and after 2 weeks of high salt (175 mM NaCl) treatment, the root elongations of IhCHS1-OE1 and IhCHS1-OE2 transgenic lines were 36.7% and 26.46% higher, respectively, than that of wild type. The above results indicate that overexpressing iris tectorum schizandrum IhCHS1 can significantly enhance the salt tolerance of transgenic arabidopsis seedlings.

5. To further understand the effect of over-expressed Iris philippica IhCHS1 on salt tolerance of transgenic Arabidopsis thaliana in maturity, transgenic and wild type seedlings grown on MS medium for 2 weeks were transferred to hydroponic conditions for 4 weeks, and then subjected to salt treatment (150 mM NaCl) with normal nutrient solution (1/4 Hogland) as a control, and after 6d treatment with 150mM NaCl, the plant mature growth phenotype was photographed and measured for flower stem height and biomass, respectively.

It was found that under normal conditions there was no significant difference in growth between wild type and transgenic IhCHS a. Thaliana, whereas after 6 days of salt treatment both transgenic lines IhCHS-OE 1 and IhCHS-OE 2 grew significantly better than wild type plants (fig. 6A), especially transgenic IhCHS a. Thaliana with a significantly higher flower stem height than wild type plants (fig. 6B). In addition, under salt stress conditions, fresh and dry weights of the aerial parts of transgenic IhCHS a were also significantly greater than those of wild-type plants (fig. 7). The above research results show that the over-expression of Iris philippinensis IhCHS1 can significantly enhance the salt tolerance of transgenic Arabidopsis thaliana in the mature period.

Example 4 analysis of accumulation Properties of jasmonate in transgenic salt-loving iris IhCHS Gene plant

1. Firstly, the T3 generation homozygote of the transgenic IhCHS gene and the wild type arabidopsis seeds are respectively sown in an MS culture medium (containing 1% of sucrose and 1% of agar) after being disinfected, and are vertically cultured in an illumination incubator after being synchronized for 3 days at 4 degrees, wherein the culture conditions are shown in the example 3. Seedlings of 2 weeks of age with consistent growth were selected and transferred to nutrient solution (1/4 Hogland) for 10 days and salt treatment (100 mM NaCl) was performed on the different lines. After NaCl treatment for 24 hours, taking a whole sample of each strain of arabidopsis thaliana, and carrying out qualitative and quantitative analysis on metabolites.

2. 50Mg of samples of Arabidopsis thaliana of different strains were weighed separately, 1000. Mu.L of an extract containing an internal standard (methanol, acetonitrile and water volume ratio=2:2:1) was added for grinding and sonication, and after centrifugation at 12000rpm for 15min at 4℃the supernatant was dried in a vacuum concentrator. Redissolving the vacuum dried extract in acetonitrile-water solution (v/v=1:1), and analyzing metabolites in the sample to be tested by using an ultra-high performance liquid chromatography tandem high resolution mass spectrometer system. The qualitative and quantitative analysis of the metabolites mainly uses raw data acquired by MassLynx V4.2 to perform data processing operations such as peak extraction, peak alignment and the like through Progenesis QI software. And carrying out identification on the basis of Progenesis QI software on line METLIN database and public database, and simultaneously carrying out theoretical fragment identification, wherein the mass number deviation of parent ions is 100ppm and the mass number deviation of fragment ions is within 50 ppm.

3. The jasmonate compounds which are accumulated in different strains and are remarkably different are screened and identified by utilizing the quantitative results of the KEGG, HMDB and LIPID MAPS databases compared with the binding metabolites.

The results of the study showed that accumulation of most compounds of the jasmonate metabolic pathway in transgenic lines overexpressing IhCHS1 showed a significant (p < 0.05) trend to increase under salt stress conditions compared to wild-type controls, including jasmonic acid (Jasmonic acid and 7-epi-Jasmonic acid) and its synthetic precursors (12-OPDA) and derivatives [ methyl jasmonate (Methyl jasmonate) and jasmonic acid- (L) -isoleucine (Jasmonoyl-L-isoleucine) ] (fig. 8). This indicates that the tectoridone-camptotheca synthase gene IhCHS has the effect of enhancing biosynthesis of jasmonate under salt stress.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims

1. A method for improving plant salt tolerance and/or jasmonic acid accumulation ability, characterized in that it comprises the following steps:

overexpressing a chalcone synthase encoding gene in the plant;

The amino acid sequence of the chalcone synthase is shown in SEQ ID NO.1.

2. The method according to claim 1, characterized in that it comprises at least one of the following features:

(1) The plants include plants of the Iridaceae family;

(2) The plants include plants of the family Cruciferae;

(3) The plant includes an iris plant;

(4) The plant includes a plant of the genus Arabidopsis;

(5) The plant includes Arabidopsis thaliana.

3. Use of a nucleic acid molecule encoding a chalcone synthase in the preparation of a product for improving plant salt tolerance and/or jasmonic acid accumulation ability, characterized in that the amino acid sequence of the chalcone synthase is shown in SEQ ID NO.1.

4. The use according to claim 3, characterized in that the improving the salt tolerance of plants and/or the ability to accumulate jasmonic acid substances comprises improving the growth ability of plants under salt stress and/or improving the ability of plants to synthesize one or more of jasmonic acid, jasmonic acid derivatives and the precursor substance 12-oxo-phytodienoic acid.

5. The application according to claim 3, characterized in that it comprises at least one of the following features:

(1) The product contains a DNA sequence or RNA sequence encoding chalcone synthase;

(2) The plant includes a plant of the Iridaceae family and/or a plant of the Cruciferae family; preferably, the plant of the Iridaceae family includes a plant of the genus Iris; the plant of the Cruciferae family includes a plant of the genus Arabidopsis; most preferably, the plant is Arabidopsis thaliana.

6. A product for cultivating salt-tolerant and/or high jasmonic acid accumulating plants, characterized in that the product contains a nucleic acid molecule encoding chalcone synthase, and the amino acid sequence of the chalcone synthase is shown in SEQ ID NO.1.

7. A method for cultivating transgenic plants, characterized in that it comprises the following steps: expressing a chalcone synthase encoding gene in a recipient plant to obtain the transgenic plant;

The transgenic plants include salt-tolerant and/or high jasmonic acid accumulation plants or plant models,

The amino acid sequence of the chalcone synthase is shown in SEQ ID NO.1.

8. The method according to claim 7, characterized in that it comprises at least one of the following features:

(1) The method specifically comprises the following steps: introducing a chalcone synthase encoding gene into a recipient plant, cultivating until a homozygous plant expressing the chalcone synthase encoding gene is obtained, thereby obtaining the transgenic plant;

9. A chalcone synthase for improving plant salt tolerance and/or jasmonic acid accumulation ability, characterized in that the amino acid sequence of the chalcone synthase is shown in SEQ ID NO.1.

10. A nucleic acid molecule encoding the chalcone synthase according to claim 9, a recombinant expression vector or a recombinant cell containing the chalcone synthase encoding gene.