Barley HvHCT1 gene, protein and application of barley HvHCT1 gene and protein in regulation and control of plant salt tolerance

Abstract

The invention belongs to the technical field of biology, and relates to a barley germination period salt-tolerant gene HvHCT1, a protein and an application of the protein in salt damage stress resistance, and the protein comprises an amino acid sequence shown as SEQ ID NO: 2. According to the method, genetic loci related to the salt tolerance of the barley in the germination period are mined through a whole genome association analysis method, then transcriptome sequencing is used for revealing barley salt tolerance differential gene expression changes after salt damage stress, and in combination with association analysis and transcriptome sequencing data, salt tolerance candidate genes in the germination period of the barley are determined. The transgenic arabidopsis thaliana is used for verification; the research provides an excellent germplasm material and a new salt-tolerant gene for barley salt-tolerant breeding research, and also provides a theoretical basis for barley salt-tolerant molecular marker-assisted breeding in the future.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a barley HvHCT1 gene, a protein and an application thereof in regulating plant salt tolerance. Background Art

[0002] Soil salinization is one of the main abiotic stresses limiting global crop production. Due to the natural environment and unreasonable human activities, the problem of land salinization is becoming more and more serious, and has become one of the important issues threatening food security and sustainable development in arid and semi-arid areas worldwide. The global saline soil area exceeds 833 million hectares, accounting for 8.7% of the earth's area.

[0003] Barley (Hordeum vulgare L.) is an annual herbaceous plant of the genus Hordeum in the Poaceae family. It is one of the main food crops in the world. It has been cultivated in my country for thousands of years and has many uses such as food, feed, medicine, and brewing. Barley has a strong adaptability to the environment and has excellent salt-alkali resistance. It is of great significance to solve the salinization of land and is a pioneer crop for the development of saline-alkali land. At the same time, Yancheng, Jiangsu is one of the four major production areas of beer barley in my country. Therefore, identifying barley salt-tolerant germplasm resources and exploring barley salt-tolerant genes are of great significance for breeding new varieties of excellent salt-tolerant barley and even more excellent salt-tolerant crops. Therefore, identifying barley salt-tolerant germplasm, exploring key salt-tolerant genes, and exploring their biological functions and molecular mechanisms are of great significance for breeding new varieties of excellent salt-tolerant barley.

[0004] At present, most of the research on plant salt tolerance is still based on model plants such as Arabidopsis and rice. Although the mechanisms of different plants in coping with salt stress are somewhat conservative, they are also very different. Research on barley salt tolerance has been relatively slow, and salt-tolerant gene resources are lacking. Barley's salt tolerance is significantly stronger than other cereal crops such as rice and wheat. It is a pioneer crop for the utilization of saline-alkali land and an excellent material for exploring key salt-tolerant genes and analyzing new mechanisms of plant salt tolerance. Therefore, it is particularly important to carry out basic and applied research on salt tolerance in barley, draw on research results in model organisms, and deeply analyze its signal transduction and molecular regulatory networks in response to salt stress. Summary of the invention

[0005] In view of the current situation that there are few studies on genes and molecular mechanisms related to salt tolerance during the germination period of barley, the present invention mines genes related to salt tolerance during the germination period of barley through whole-genome association analysis, and then uses transcriptome sequencing to identify differentially expressed genes related to salt tolerance during the germination period of barley. Finally, the association analysis and transcriptome sequencing are combined to accurately and quickly screen and identify candidate genes for salt tolerance during the germination period of barley, and the transgenic Arabidopsis technology is used to further verify the functions of the candidate genes. Molecular markers are designed for the identified barley salt tolerance genes during the germination period, which provides a certain reference for the later molecular marker-assisted breeding (MAS) selection.

[0006] The technical solution provided by the present invention is as follows:

[0007] A barley HvHCT1 protein, wherein the protein is the protein of a) or b) below:

[0008] a) a protein consisting of the amino acid sequence shown in SEQ ID No.2;

[0009] b) A protein derived from SEQ ID No. 2 which is related to the root development of loquat and in which one or more amino acid residues are substituted and / or added and / or deleted.

[0010] A barley HvHCT1 gene, which encodes the above protein.

[0011] Furthermore, the gene sequence is shown in SEQ ID NO:1.

[0012] The present invention also provides an expression box, a recombinant vector and a recombinant microorganism containing the gene.

[0013] Application of the above protein or gene or expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the above gene in regulating plant salt tolerance.

[0014] Furthermore, the regulation of plant salt tolerance is regulation of barley salt tolerance during germination.

[0015] Furthermore, the regulation of plant salt tolerance is to increase plant height, chlorophyll content, stem and leaf fresh weight, stem and leaf dry weight and root length of plants under salt stress conditions.

[0016] Application of the above protein or the above gene or an expression box, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the above gene in cultivating salt-tolerant varieties of barley or Arabidopsis.

[0017] A method for cultivating salt-tolerant plant varieties, wherein the above gene is transferred into a target plant to obtain a transgenic plant; the salt tolerance of the transgenic plant is higher than that of the target plant; the plant is barley or Arabidopsis thaliana.

[0018] Furthermore, the amplification primers for the gene are:

[0019] P1 forward primer: 5′- GGTGACGTTCGAGATGCAGA-3′;

[0020] P2 reverse primer: 5'-AATAGGGGCACGAAGGCATC-3'.

[0021] Furthermore, the target gene was connected to the pDONR221 vector to construct an entry vector, and then the entry vector was connected to the pB2GW7 vector to construct an overexpression vector containing the target gene.

[0022] Furthermore, it is characterized in that the above gene is introduced into plants for expression using Agrobacterium-mediated genetic transformation.

[0023] A method for rapidly mining salt-tolerance genes in barley during germination using genome-wide association analysis, comprising the following steps:

[0024] (1) Rapid screening and identification of salt-tolerant resources during the germination period of barley: 250 core barley germplasm resources from different regions and types (including 148 two-row barley and 102 six-row barley). They came from 19 countries, with the largest number of germplasms from China (172), followed by Japan and Germany. Artificial salt damage was simulated in the laboratory, and the barley salt tolerance concentration screening experiment showed that 0.8% NaCl concentration was the appropriate concentration for screening salt-tolerant germplasm. 250 barley germplasm resources were germinated and cultured in pure water with 0.8% NaCl. Under suitable conditions, after one week of control and treatment culture, the relevant traits of barley seeds, such as germination rate, root length, bud length, coleoptile length, root weight, and bud weight, were examined.

[0025] (2) Determination of loci associated with salt tolerance during barley germination:

[0026] Using GEMMA software, a mixed linear model (MLM) was used to conduct genome-wide association analysis on salt tolerance-related traits during germination combined with molecular marker data. 10 (P value) ≥4.0 was the threshold value, and a total of 188 significantly associated SNP sites were mined for the six salt tolerance traits, of which 11 were stable sites that were associated with multiple traits at the same time, indicating that the locus gene may have one cause and multiple effects. The locus located near 573.35Mb on chromosome 5 was detected by multiple traits at the same time, and had the highest genetic efficiency, indicating that it was closely related to barley salt tolerance.

[0027] Another method for determining the phenotype and physiological indexes of barley salt-tolerant materials includes the following steps:

[0028] Based on the salt tolerance identification of 250 core barley germplasms, the extremely salt-tolerant material six-angled barley (LLZDM, Jiangsu local variety) and the extremely sensitive material Naso Nijo (NN, Japanese variety) were selected for further analysis, and the root length, stem length, root fresh weight, and stem fresh weight under control and salt stress were investigated; Through the identification of salt tolerance of germplasm resources at different periods, two extreme difference materials Naso Nijo (extremely sensitive) and six-angled barley (extremely salt-tolerant) were selected. Salt stress during the germination period seriously affected the growth and development of barley, and the traits such as bud length, root length, bud fresh weight, and root fresh weight all decreased significantly, but the decrease in six-angled barley was much smaller than that in Naso Nijo, indicating that six-angled barley has stronger salt tolerance. The increase was significantly higher than that of the sensitive material NN, while the increase in malondialdehyde, superoxide anion, and hydrogen peroxide content was higher in NN than in LLZDM. The content of malondialdehyde reflected the degree of damage to the cell membrane of the experimental materials, and superoxide anions and hydrogen peroxide reflected the degree of damage to the leaves and roots of the plants. The degree of damage to NN after salt stress was higher than that to LLZDM.

[0029] Another method for mining differentially expressed genes for salt tolerance during barley germination by transcriptome sequencing analysis includes the following steps:

[0030] (1) After salt damage treatment of the extremely salt-tolerant material Hordeum vulgare and the extremely sensitive material Naso Nijo, transcriptome sequencing analysis was performed on the germinated seeds. After salt damage stress, the number of differentially expressed genes in the salt-tolerant type Hordeum vulgare was 1776, and the number of differentially expressed genes in the sensitive type Naso Nijo was 839. The number of up-regulated differentially expressed genes in the salt-tolerant type barley after salt stress was 962, and the number of down-regulated differentially expressed genes was 813; the number of up-regulated differentially expressed genes in the sensitive type barley after salt stress was 464, and the number of down-regulated differentially expressed genes was 375, indicating that after salt stress, the number of up-regulated and down-regulated differentially expressed genes in the salt-tolerant type barley was higher than that in the sensitive type.

[0031] (2) GO functional analysis and KEGG enrichment analysis of differentially expressed genes

[0032] To further study the regulatory pathways of differentially expressed genes, GO and KEGG enrichment analysis was performed on the differentially expressed genes. The metabolic pathways significantly enriched in salt-tolerant materials include: phenylpropanoid biosynthesis, endoplasmic reticulum protein processing, exopolysaccharide biosynthesis, betaine biosynthesis, stilbene compounds, biosynthesis of diarylheptane and gingerol, ion transport channels and other metabolic pathways. Many related genes in the energy metabolism pathway are closely related to plant stress. For example, metabolic pathways such as antioxidant enzymes and glycolysis are significantly enriched in salt-tolerant materials, indicating that the genes in these pathways play an important regulatory role in responding to salt stress.

[0033] (3) Heat map analysis of salt tolerance differential gene clustering

[0034] Phenylpropanoid biosynthesis, antioxidant enzymes, hormones, and transcription factors play an important role in plant salt stress. By comparing transcriptome data, cluster heat map analysis was performed on the differentially expressed genes in these three metabolic pathways. After salt stress, the HCT, CHS, and CHI genes in the phenylpropanoid biosynthesis regulatory pathway were significantly upregulated in LLZDM, while the upregulation was significantly smaller in the sensitive material NN. At the same time, in the hormone regulatory pathway, it was found that genes such as ABI, GA, and SAUR responded to salt stress by regulating signals such as ABA and auxin, and transcription factors such as AP2 / ERF, HSF, MADS, and PLATZ were also actively involved in the regulation of barley salt tolerance.

[0035] (4) Mining candidate genes for salt tolerance

[0036] Based on the results of genome-wide association analysis and combined with transcriptome sequencing data, candidate genes for salt tolerance were screened within the 100 kb range above and below the significant association sites, and a total of 15 candidate genes for salt tolerance were identified. These genes are closely related to ion transport, membrane-associated proteins, ATP synthesis, peroxidase, etc., which can control the Na in the cell membrane. + Ion transport, removal of reactive oxygen accumulated in plants, promotion of auxin synthesis, etc. Coumaroyl-CoA ligase located on chromosome 5 is an important intermediate metabolite in the phenylalanine metabolic pathway. The up-regulated expression of this gene promotes the synthesis of substances such as flavonoids. Flavonoids further improve the salt-alkali tolerance of plants through antioxidant enzymes, plant hormones, transcription factors, etc.

[0037] A determination of a salt-tolerance gene for barley and its breeding application, comprising the following steps:

[0038] (1) Identification and acquisition of candidate genes for salt tolerance in barley

[0039] The whole genome association analysis combined with transcriptome sequencing analysis located the barley salt tolerance candidate gene near 573.35Mb on chromosome 5. Comparison with the barley reference genome and combined with transcriptome gene differential expression analysis found that the HCT1 gene near this region was significantly upregulated in the salt-tolerant material Hordeum vulgare after salt damage treatment, while the upregulation was smaller in the sensitive material NasoNijo, and was determined to be a barley salt tolerance candidate gene. Using Hordeum vulgare seedlings as the material, the total RNA of the plant was extracted, and the gene sequence of HvHCT1 was amplified by RT-PCR. The amplified product was then connected to the pGEM-Teasy vector, and the clone with the target gene was obtained by sequencing.

[0040] (2) Construction of plant expression vector and genetic transformation: Invitrogen's Gateway technology was used to construct a barley HvHCT1 gene overexpression vector. The target gene was connected to the pDONR221 vector using BP Clonase™ enzyme to construct an entry vector, and then the entry vector was connected to the pB2GW7 vector using LR Clonase™ enzyme to construct an overexpression vector containing the target gene. The overexpression vector containing the target gene was introduced into Agrobacterium Gv3101 by liquid nitrogen freeze-thaw method. The HvHCT1 coding sequence was introduced into plants such as Arabidopsis for expression using Agrobacterium-mediated genetic transformation. Positive transgenic plants were screened by antibiotic screening, RT-PCR, etc.

[0041] (3) Analysis of salt tolerance of transgenic plants: Arabidopsis seeds (wild type and transgenic strains) in the germination stage were placed on a culture medium containing 0.8% NaCl, and then transferred to a matrix for normal growth and development. The phenotypes of transgenic plants and wild type plants were observed, so as to screen out transgenic plants with obvious resistance to salt damage.

[0042] Beneficial Effects

[0043] Genome-wide association studies (GWAS) are based on linkage disequilibrium and can combine phenotypic differences in natural population traits with gene polymorphisms at the level of the crop genome, thereby identifying molecular markers related to phenotypic differences. GWAS is different from traditional QTL positioning. GWAS does not require the construction of a special mapping population, and can directly screen out genes that are significantly associated with the target trait. Transcriptome sequencing (RNA-seq) technology can obtain specific gene sequences and gene expression in a timely manner. The joint analysis of GWAS and RNA-seq can more efficiently identify stress-related candidate genes.

[0044] In order to screen out barley germplasm adapted to saline-alkali land cultivation, the present invention explores the germination characteristics of different barley germplasms and their effects on the salt tolerance of seedlings by treating them with salt damage during the seed germination period, and uses GWAS to detect SNP sites related to barley salt tolerance traits, and then combines transcriptome sequencing (RNA-seq) technology to quickly and accurately mine candidate genes for barley salt tolerance. Flavonoids are widely considered to be secondary metabolites in the plant defense system. Previous studies have emphasized the antioxidant and free radical scavenging properties of flavonoids, such as intermediate metabolites such as neohesperidin and quercetin. There have been no reports on the effects of coumaroyl-CoA, an intermediate metabolite in the flavonoid metabolic pathway, on plant salt tolerance. This study is of great significance for exploring the molecular mechanism of salt tolerance in wheat crops and molecular marker-assisted breeding of new salt-tolerant barley varieties.

[0045] The invention selects 250 natural populations of barley varieties with a wide range of sources and no direct genetic relationship as materials, uses simplified genome sequencing to obtain 106131 high-quality SNP markers, screens and identifies the salt tolerance of barley germplasm resources during the germination period in a laboratory incubator, combines the SNP markers, and uses a whole genome association analysis method to obtain molecular marker sites closely associated with the salt tolerance of barley during the germination period; uses transcriptome sequencing to perform transcriptome sequencing analysis on seeds of salt-sensitive materials and salt-tolerant materials during the germination period, discovers differentially expressed genes associated with salt tolerance during the germination period, and finally determines a barley salt tolerance candidate gene HvHCT1 during the germination period by combining association analysis with transcriptome analysis, and further verifies the function of the gene by using Arabidopsis thaliana transgenic plants, and no report has been seen on the research of the gene in terms of salt tolerance. Under salt stress, the plant height, chlorophyll content, stem and leaf fresh weight, stem and leaf dry weight, root length and other traits of the transgenic materials increased by more than 50%. This study provided excellent germplasm materials and excellent alleles for barley salt-tolerant breeding research, which can ensure the stability of wheat crop yields under extreme weather conditions such as salt damage.

[0046] The present invention uses whole genome association analysis combined with transcriptome sequencing to rapidly and accurately identify salt-tolerant genes in barley during the germination period, and verifies their functions through transgenic technology, laying a foundation for the future breeding of new salt-tolerant barley varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Manhattan plot of genome-wide association analysis using salt tolerance traits of barley during germination; A: relative germination rate; B: relative shoot length association analysis plot; C: relative coleoptile association analysis plot; D: relative root length association analysis plot; E: relative shoot fresh weight association analysis plot; F: relative root fresh weight association analysis plot.

[0048] Figure 2 Phenotypic analysis of extremely salt-tolerant and salt-sensitive materials under salt stress; A, B: phenotypic differences between two materials; C: comparative analysis of shoot height; D: comparative analysis of root length; E: comparative analysis of root fresh weight; F: comparative analysis of seedling fresh weight; Note: LLZDM: Hordeum vulgare; NN: Naso Nijo.

[0049] Figure 3 Analysis of physiological and biochemical indices of extremely salt-tolerant and salt-sensitive materials under salt stress; AH: changes in physiological indices under salt stress; Note: AB: NBT staining; CD: DAB staining; E: superoxide dismutase (SOD) activity, F: peroxidase (POD) content, G: catalase (CAT) content, H: malondialdehyde (MDA) content.

[0050] Figure 4 Transcriptome analysis of salt-tolerant and salt-sensitive materials under salt stress treatment;

[0051] A: PCA principal component analysis; B: Comparison of the number of differentially expressed genes between the two materials; C: Cluster analysis of differentially expressed genes in Hordeum vulgare; D: Cluster analysis of differentially expressed genes in Naso Nijo. Note: A: PCA cluster analysis of sequencing of different materials; B: Venn diagram of two barley varieties after salt stress; C: KEGG enrichment analysis of differentially expressed genes; D: Cluster heat map of differentially expressed genes of antioxidant enzymes. HCT, shikimate / quinate hydroxycinnamoyltransferase; CHS, chalcone synthase; CHI, chalcone isomerase.

[0052] Figure 5 Gene expression analysis of barley HvHCT1 gene among different materials.

[0053] Figure 6 Phenotypic analysis of salt tolerance of Arabidopsis transgenic and non-transgenic strains. A: Differences between wild-type and transgenic strains at the germination stage in the culture medium after salt stress; B: Differences between wild-type and transgenic strains at the seedling stage in the culture medium after salt stress; C: Growth under control conditions; D: Growth comparison after salt stress. DETAILED DESCRIPTION

[0054] Example 1 Genome-wide association analysis of salt tolerance in barley during germination

[0055] The present invention is further described below with reference to the examples. The experimental methods in the following examples without specifying specific conditions are generally carried out according to the well-known means in the art.

[0056] (1) Acquisition of experimental materials and their salt tolerance identification phenotypic data:

[0057] Experimental materials: A total of 250 core barley germplasms were collected, from 19 countries and with a wide distribution range. Among them, the number of germplasms from China is the largest, with more than 170, accounting for 68.8%. The second largest number of germplasms comes from Japan and Germany, accounting for 5.2% and 4.8% respectively. At the same time, the barley germplasms collected this time have two ridge types, namely two-row barley and six-row barley. There are 148 two-row barley and 102 six-row barley.

[0058] Salt damage treatment method: In order to identify the salt tolerance of barley germplasm resources, we used the local main barley variety Yanmai No. 7 as the experimental material, set different NaCl concentrations of 0, 0.5%, 0.8%, 1.2%, 1.5%, and 2%, and examined the germination rate, seedling length, coleoptile length, seedling fresh weight, root length, and root fresh weight. The results showed that when the salt concentration was 0.5% and 0.8%, the plant phenotype decreased significantly compared with the control; when the salt concentration was greater than 1.2%, the germination rate, plant seedling height, root length, etc. were significantly inhibited, and most seeds could not germinate. Therefore, 0.8% NaCl concentration is a suitable concentration for screening salt-tolerant germplasm. In order to further identify barley salt-tolerant germplasm, salt damage was artificially simulated in the laboratory, and the salt tolerance of 250 barley germplasm resources during the germination period was screened and identified. By comparing the germination rate, root and shoot growth length and other traits of barley seed resources after salt damage, it was found that there were obvious differences between the control group and the treatment group, and there were also significant differences between different genotypes.

[0059] (2) Genome-wide association analysis of salt tolerance during barley germination:

[0060] A genome-wide association analysis was performed on barley salt tolerance-related traits, and a total of 188 significantly associated SNP loci were discovered for the six salt tolerance traits. Among them, 58 SNP loci were detected to be significantly associated with the germination rate of the treatment group, and 27 SNP loci were significantly associated with the relative germination rate ( Figure 1 -A). Among them, 70 SNP loci were found to be significantly associated with salt tolerance in barley seedlings ( Figure 1 -B), 11 SNP loci were detected that were significantly associated with relative bud length in barley ( Figure 1 -B), there are 45 SNP loci significantly associated with coleoptile length ( Figure 1 -C), there are 14 SNPs associated with relative bud fresh weight ( Figure 1 -E). The analysis mined genes related to barley salt-tolerant root traits, among which 33 SNP loci were found to be significantly associated with barley root salt tolerance, 16 SNP loci were detected to be significantly associated with relative root length, and 17 SNP loci were detected to be significantly associated with relative root fresh weight. Figure 1 -F). Among the 188 SNP sites detected, 11 were stable sites, which were associated with multiple traits at the same time, indicating that the gene at this site may have multiple effects. The SNP site near 573.35Mb on chromosome 5 was detected many times and was a stable site closely related to barley salt tolerance.

[0061] Example 2 A method for mining differentially expressed genes for salt tolerance during barley germination by transcriptome sequencing analysis comprises the following steps:

[0062] (1) Through the identification of salt tolerance of germplasm resources at different stages, two extreme difference materials, NasoNijo (extremely sensitive) and six-angled barley (extremely salt-tolerant) were screened out (Figure 2-A). Salt stress during the germination period seriously affected the growth and development of barley. The traits such as shoot length, root length, shoot fresh weight, and root fresh weight all decreased significantly, but the decline of six-angled barley was much smaller than that of NasoNijo, indicating that six-angled barley has stronger salt tolerance ( Figure 2 AF); the change range of chlorophyll content in salt-tolerant material LLZDM after salt stress was significantly lower than that in sensitive material NN. Before salt stress, there was no significant difference in antioxidant enzyme activity and malondialdehyde content between the two materials; after salt stress, the antioxidant enzyme activity and malondialdehyde content of the two materials increased significantly, but the increase range of POD, CAT, and SOD content in LLZDM barley was significantly higher than that in sensitive material NN ( Figure 3 EG), while the increase in malondialdehyde, superoxide anion, and hydrogen peroxide content was higher in NN than in LLZDM ( Figure 3 AD, H). The malondialdehyde content reflects the degree of damage to the cell membrane of the experimental materials, and the superoxide anion and hydrogen peroxide reflect the degree of damage to the leaves and roots of the plants. The degree of damage to NN after salt stress is higher than that to LLZDM.

[0063] (2) Transcriptome sequencing analysis of germinated seeds.

[0064] Transcriptome sequencing was performed on two salt-tolerant materials, and PCA principal component analysis was performed on the sequencing results. The green line frame represents the treatment group of salt-tolerant barley, the yellow line frame represents the treatment group of sensitive barley, the red line frame represents the control group of sensitive barley, and the blue line frame represents the treatment group of salt-tolerant barley. Three replicates of the same material were clustered into one category, indicating that the sequencing data of this experiment is relatively reliable and is conducive to further analysis later ( Figure 4 -A). After salt stress, the number of differentially expressed genes in salt-tolerant barley was 1776, and the number of differentially expressed genes in sensitive barley was 839. After salt stress, the number of up-regulated differentially expressed genes in salt-tolerant barley was 962, and the number of down-regulated differentially expressed genes was 813; after salt stress, the number of up-regulated differentially expressed genes in sensitive barley was 464, and the number of down-regulated differentially expressed genes was 375, indicating that after salt stress, the number of up-regulated and down-regulated differentially expressed genes in salt-tolerant barley was higher than that in sensitive barley ( Figure 4 -B).

[0065] (3) GO functional analysis and KEGG enrichment analysis of differentially expressed genes

[0066] To further study the regulatory pathways of differentially expressed genes, GO and KEGG enrichment analysis was performed on the differentially expressed genes. The metabolic pathways significantly enriched in salt-tolerant materials include: phenylpropanoid biosynthesis, endoplasmic reticulum protein processing, exopolysaccharide biosynthesis, betaine biosynthesis, stilbene compounds, biosynthesis of diarylheptane and gingerol, ion transport channels and other metabolic pathways. Many related genes in the energy metabolism pathway are closely related to plant adverse stress. For example, metabolic pathways such as antioxidant enzymes and glycolysis are significantly enriched in salt-tolerant materials, indicating that the genes in these pathways play an important regulatory role in responding to salt stress ( Figure 4 -C).

[0067] (4) Cluster heat map analysis of salt tolerance differentially expressed genes

[0068] Phenylpropanoid biosynthesis, antioxidant enzymes, and hormones play an important role in plant salt stress. By comparing transcriptome data, cluster heat map analysis was performed on the differentially expressed genes in these three metabolic pathways. After salt stress, the HCT, CHS, and CHI genes in the phenylpropanoid biosynthesis regulatory pathway were significantly upregulated in LLZDM, while the upregulation was significantly smaller in the sensitive material NN ( Figure 4 -D; Figure 5 ). At the same time, in the hormone regulation pathway, it was found that genes such as ABI, GA, and SAUR responded to salt stress by regulating signals such as ABA and auxin, and transcription factors such as AP2 / ERF, HSF, MADS, and PLATZ also actively participated in the regulation of barley salt tolerance.

[0069] Example 3 Determination of candidate genes for salt tolerance during the germination period of barley and their application in breeding

[0070] (1) Cloning of the barley salt tolerance gene HvHCT1

[0071] The whole genome association analysis combined with transcriptome sequencing analysis located the barley salt tolerance candidate gene near 573.35Mb on chromosome 5. Comparison with the barley reference genome and combined with transcriptome gene differential expression analysis found that the HvHCT1 gene near this region was significantly upregulated in the salt-tolerant material Hordeum vulgare after salt damage treatment, but the differential expression was not obvious in the sensitive material NasoNijo, and it was determined to be a barley salt tolerance candidate gene. Using Hordeum vulgare seedlings as the material, the total RNA of the plant was extracted, and the gene sequence of HvHCT1 was amplified by RT-PCR. The amplified product was then connected to the pGEM-Teasy vector, and the clone with the target gene was obtained by sequencing.

[0072] Specific primers P1 forward primer: 5'- GGTGACGTTCGAGATGCAGA-3' (SEQ ID NO: 3) and P2 reverse primer: 5'- AATAGGGGCACGAAGGCATC-3' (SEQ ID NO: 4) were designed, and total RNA was extracted from the salt-tolerant material Hordeum vulgare seedlings using an RNA extraction kit, and reverse transcribed to synthesize cDNA. The CDS sequence of the barley salt-tolerance gene shown in SEQ ID NO: 1 was amplified from the cDNA obtained by reverse transcription of RNA using the above primers P1 and P2. The full length of the CDS sequence of the gene HvHCT1 is 1289 bp.

[0073] (2) Plant expression vector construction and genetic transformation

[0074] The overexpression vector of the barley HvMADS1 gene was constructed using the Gateway technology of Invitrogen. The target gene was connected to the pDONR221 vector using BPClonase™ enzyme to construct an entry vector, and then the entry vector was connected to the pB2GW7 vector using LR Clonase™ enzyme to construct an overexpression vector containing the target gene. The overexpression vector containing the target gene was introduced into Agrobacterium Gv3101 by liquid nitrogen freeze-thaw method. Arabidopsis was transformed by inflorescence infection method using Agrobacterium-mediated genetic transformation method, and the HvHCT1 coding sequence was introduced into Arabidopsis and other plants for expression. Positive transgenic plants were screened by antibiotic screening, RT-PCR, etc.

[0075] (3) Analysis of salt tolerance of transgenic plants

[0076] In order to further analyze the biological function of barley salt tolerance gene HvHCT1, we overexpressed the gene in Arabidopsis and intended to verify the biological function of gene HvHCT1 in salt stress regulation from the phenotypic characteristics of transgenic plants. The specific experimental steps are as follows: Among the T3 positive transgenic lines obtained by transforming Arabidopsis with HvHCT1 gene in the early screening, seeds of three lines were randomly selected for subsequent experiments. When 0.8% NaCl was added to the culture medium, the germination rate of transgenic lines was significantly increased compared with the wild type, from 75% to 90%. The plant height and root length at the seedling stage were also significantly higher in transgenic lines than in the wild type. After culturing on 1 / 2MS culture medium until the 4-leaf stage, the seedlings were transplanted into pots filled with nutrient soil and cultured in an artificial climate chamber for 30 days, and then treated with 0.8% NaCl for salt damage. After two weeks of stress, the difference in salt tolerance between transgenic materials and wild-type materials was observed. Under salt stress, the plant height, chlorophyll content, stem and leaf fresh weight, stem and leaf dry weight, and root length of the wild-type material decreased by 40.09%, 57.56%, 61.80%, 60.01%, and 65.87%, respectively, compared with the control; while the transgenic material only decreased by 12.18%, 18.32%, 26.11%, 36.25%, and 38.23%. After salt stress, the salt tolerance of the transgenic strains was significantly stronger than that of the non-transgenic materials (such as Figure 6 as shown).

[0077] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

[0078] SEQ ID NO: 1:

[0079]

[0080] SEQ ID NO:2:

[0081] XSSKLVKPAYSAGDAPMPATEYIPLSIFDRVTFEMQMAIIYAYAPPAPSTASIEKGLATVLAQYRGFAGQLGESPDGTPSVILNDRGARLFEASVDADLVDMAPSKPTPELLKLHPDLEAEHREVVLLQLTRFRCGSLAVGFTSNHVVADGHATSNFLVAWGRATRGLPMGLPPVHHHKELFKPRSSPRVEHDHRNREYYLPSPTDVVGHHGDAADNIVIHKAQFTKDFIAGLRAKASEGRGRPFSRFETILAHLWRTMTRARDLSPEETSKIRLSVDGRHRLGQPAEYFGNMVLWAFPRATVGDLLNRPLKHAAQVIHDEVARVDAAYFQSFVDFASSGAAEKEGLARSAVCKDAHCPDVEVDSWLTFPFYELDFGTGSPSYFMPAFFPTEGMLFLAPSNLGDGSVDAFVPLFQENLQAFKECCYSME*。

Claims

1. A barley HvHCT1 protein, characterized in that The protein is the protein of a) or b) below: a) a protein consisting of the amino acid sequence shown in SEQ ID No.2; b) A protein derived from SEQ ID No. 2 which is related to the root development of loquat and in which one or more amino acid residues are substituted and / or added and / or deleted.

2. A barley HvHCT1 gene, characterized in that The gene encodes the protein according to claim 1.

3. The gene according to claim 2, characterized in that The gene sequence is shown in SEQ ID NO:

1.

4. An expression cassette, recombinant vector, or recombinant microorganism containing the gene according to claim 2 or 3.

5. Use of the protein according to claim 1 or the gene according to claim 2 or 3 or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene according to claim 2 or 3 in regulating plant salt tolerance.

6. The use according to claim 5, characterized in that: The method of regulating plant salt tolerance is regulating barley salt tolerance during germination.

7. Use of the protein according to claim 1 or the gene according to claim 2 or 3 or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene according to claim 2 or 3 in breeding salt-tolerant plant varieties of barley or Arabidopsis.

8. A method for cultivating salt-tolerant plant varieties, characterized in that: The gene described in claim 2 or 3 is transferred into a target plant to obtain a transgenic plant; the salt tolerance of the transgenic plant is higher than that of the target plant; the plant is barley or Arabidopsis thaliana.

9. The method for cultivating salt-tolerant plant varieties according to claim 8, characterized in that: The amplification primers for the gene are: P1 forward primer: 5′- GGTGACGTTCGAGATGCAGA-3′; P2 reverse primer: 5'-AATAGGGGCACGAAGGCATC-3'.

10. The method for cultivating salt-tolerant plant varieties according to claim 8, characterized in that: The target gene was connected to the pDONR221 vector to construct an entry vector, and then the entry vector was connected to the pB2GW7 vector to construct an overexpression vector containing the target gene.

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