Gene HvERF62 for regulating and controlling moisture resistance of barley and application of gene HvERF62

Abstract

The invention belongs to the field of molecular genetics, and particularly relates to an ethylene response transcription factor HvERF62 for regulating and controlling barley moisture resistance and application of the ethylene response transcription factor HvERF62 in molecular marker-assisted selective breeding. According to the invention, a site which obviously affects the moisture resistance of barley is identified through whole genome association analysis, a candidate gene is predicted, and the moisture resistance function of the candidate gene HvERF62 is further verified by adopting a CRISPR-Cas9 gene editing method. The function of HvERF62 for regulating and controlling the barley moisture resistance is defined through a gene editing technology, the InDel molecular marker for barley moisture resistance detection is developed, 3% agarose gel is used for detection of the molecular marker, expression is stable, the method is simple, barley moisture resistance can be screened in the early stage, and the molecular marker has high application value in barley moisture resistance breeding.

Description

Technical Field

[0001] The invention relates to the field of molecular genetics, and in particular to a gene HvERF62 for regulating barley moisture tolerance and an application thereof. Background Art

[0002] Wet damage is a major natural disaster in many countries in the world. In recent years, with the intensification of global climate anomalies and the serious damage to the ecosystem, floods have continued in local areas, and soil flooding is extremely common. According to statistics from the Food and Agriculture Organization of the United Nations in 2017, between 2006 and 2016, agricultural economic losses caused by wet damage worldwide amounted to billions of dollars, accounting for about 2 / 3 of all losses. my country is a country where floods and wet disasters frequently occur. According to statistics, about 2 / 3 of my country's land area has varying degrees of wet damage, and the most serious is the Huanghuai Plain and the middle and lower reaches of the Yangtze River, accounting for more than 3 / 4 of the affected area in the country. The barley planting area in Hubei, Anhui, Jiangsu, Zhejiang, Shanghai and other provinces and cities in the middle and lower reaches of the Yangtze River is the largest, accounting for about 1 / 2 of the national barley planting area and 2 / 3 of the total barley production in the country. Barley is often damaged by wet damage due to heavy rains, which seriously affects barley production.

[0003] Barley is a crop that is sensitive to moisture damage. Under moisture stress, excessive water hinders the transmission of oxygen in the air to the plant, causing the plant root system to be in a hypoxic or even anaerobic state, which directly affects the normal physiological activities of the root system, leading to reduced yields or even no harvest. The effects of moisture stress on plant phenotypic traits are mainly manifested in leaf wilting and drooping, chlorosis, yellowing and even rotting, inhibition of new leaf formation, reduction in total leaf area, blackening and rotting of the root system, induction of adventitious roots and formation of rhizosphere aerenchyma. If moisture damage occurs during the germination or seedling establishment period of barley, it will greatly reduce the seed germination rate and seedling survival rate. If it occurs in the middle and late stages of barley growth, it will greatly affect barley growth and reduce its yield and quality.

[0004] Numerous studies have shown that the formation of aerenchyma and adventitious roots after flooding is related to the accumulation of ethylene, and ethylene response factor (ERF) plays an important role. Rice can regulate the expression of ERFs genes through ethylene signals, promote or inhibit internode elongation, and adapt to different types of moisture stress. Overexpression of ERF genes in crops such as Arabidopsis, wheat, and rapeseed can improve moisture tolerance. In barley, there has been no research report confirming the involvement of ERF genes in barley moisture tolerance through transgenic means.

[0005] Plant moisture tolerance research is the key to improving crop moisture tolerance to cope with increasingly severe extreme climates and large-scale production management. In recent years, research using common wild rice and Arabidopsis as model plants has made breakthrough progress, which has deepened our understanding of the response mechanism to moisture stress. However, few moisture tolerance genes have been cloned in barley. Therefore, we urgently need to discover key genes related to barley moisture tolerance and provide genetic resources and molecular markers for barley moisture tolerance breeding. Summary of the invention

[0006] The purpose of the present invention is to provide a gene HvERF62 and its excellent haplotypes that can regulate the moisture tolerance of barley, and to develop InDel molecular markers based on its excellent haplotypes, so as to provide gene resources and molecular markers for the identification of moisture-tolerant barley materials and variety breeding. The present invention identifies the key moisture-tolerant gene HvERF62 through whole genome association analysis and transgenic technology. The moisture tolerance of the HvERF62 knockout mutant is significantly reduced, which is the first time to prove that HvERF62 positively regulates the moisture tolerance of barley. Analysis of its coding region sequence in 334 barley varieties found that HvERF62 mainly has 3 haplotypes, and the haplotype HvERF62-3 has a premature termination of the amino acid encoded by the base deletion, so the moisture tolerance is significantly reduced. The InDel molecular markers developed based on the difference can be used to screen moisture-tolerant barley varieties or lines. The present invention provides important gene resources and molecular markers for improving the moisture tolerance of barley, and has important application value for barley moisture tolerance breeding.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] A gene HvERF62 regulating barley moisture tolerance, the nucleotide sequence of the barley moisture tolerance gene HvERF62 is shown in SEQ ID NO.1, and the full length of the gene CDS is 987 bp.

[0009] Furthermore, the amino acid sequence of the barley ethylene-responsive transcription factor HvERF62 is shown in SEQ ID NO. 2. The protein consists of 328 amino acid residues, and the corresponding amino acid sequence is shown in SEQ ID NO. 2. The protein has a molecular weight of 35.03 KDa, an isoelectric point of 5.64, and functional domain prediction analysis shows that it contains 1 AP2 / ERF functional domain.

[0010] The present invention also provides the use of the above gene or protein in regulating barley moisture tolerance.

[0011] The present invention also provides a method for regulating moisture tolerance of barley, which comprises regulating the content and / or activity of the above protein in the target plant to obtain a mutant plant.

[0012] Furthermore, CRISPR / Cas9 gene editing technology was used to construct a recombinant plasmid pCas9-HvERF62 expressing the Cas9 protein gene and the sgRNA gene, and transgenic plants were cultured.

[0013] Furthermore, the target site primer sequences of sgRNA are shown in SEQ ID No.3 and SEQ ID No.4.

[0014] Furthermore, the primer sequences for detecting transgenic plants are shown in SEQ ID No.5 and SEQ ID No.6.

[0015] The barley ethylene response transcription factor HvERF62 has natural variations in barley, including three haplotypes: Hap1, Hap2 and Hap3.

[0016] Furthermore, the correlation between HvERF62 haplotype and chlorophyll content under moisture stress was analyzed, and the superior haplotype of HvERF62 was identified, which can be applied to improve the moisture tolerance of barley.

[0017] Furthermore, referring to the sequence differences between haplotypes, InDel molecular markers are developed for identification of barley moisture tolerance varieties and molecular marker-assisted selection breeding. According to the application of the InDel molecular markers, the primers or the detection kit in detecting or identifying barley moisture tolerance breeding.

[0018] The present invention also provides an InDel molecular marker for regulating barley moisture tolerance. The sequences of the InDel molecular marker primers for regulating barley moisture tolerance are shown in SEQ ID No.7 and SEQ ID No.8.

[0019] Furthermore, the sequences of the PCR products amplified by the InDel molecular markers SEQ ID No.7 and SEQ ID No.8 in the natural population are shown in SEQ ID No.9 and SEQ ID No.10; from the 5' end, the 39th to 42nd bases of the SEQ ID NO.9 sequence are CCCA, the 57th to 59th bases are GTC, the 99th to 116th bases are CTGCGGGCGGAGGAGGAC, and the 180th to 181st bases are TA, and the genotyping is Hap1 / 2 type, which is a moisture damage-resistant material; while the nucleotide sequence shown in SEQ ID NO.10 has base deletions at the above nucleotide positions, and the genotyping is Hap3 type, which is a moisture damage-sensitive material.

[0020] The present invention also provides the use of the above-mentioned InDel molecular marker for regulating barley moisture tolerance in barley moisture tolerance variety screening and molecular marker-assisted selection breeding.

[0021] Furthermore, the reaction procedure of the PCR amplification is 94°C for 5 min; 94°C for 30 sec, 55°C for 30 sec, 72°C for 1.5 min, 32 cycles; 72°C for 10 min.

[0022] Furthermore, the reaction system of the PCR amplification is 5 μL of 2×PCR MasterMix, 0.5 μL of forward primer, 0.5 μL of reverse primer, 0.5 μL of genomic DNA, and sterile deionized water is added to 10 μL.

[0023] Furthermore, the length of the PCR amplification product was detected and analyzed by 3% agarose gel for genotyping of the sample to be tested.

[0024] Beneficial Effects

[0025] The invention provides a new gene HvERF62 capable of regulating barley moisture tolerance. The gene HvERF62 positively regulates barley moisture tolerance and provides gene resources for barley moisture tolerance breeding.

[0026] HvERF62 mainly exists in 334 barley varieties. Among them, haplotype 3 has premature termination of amino acid coding due to base deletion, thus significantly reducing moisture tolerance. The present invention screened out the moisture-tolerant excellent haplotype Hap1 in the natural barley population, which can be applied to the genetic improvement of barley moisture tolerance.

[0027] The invention provides an InDel molecular marker, which provides a tool for barley moisture-tolerant variety screening and molecular marker-assisted selection breeding.

[0028] The present invention clarifies the function of HvERF62 in regulating barley moisture tolerance through gene editing technology, and develops InDel molecular markers for detecting barley moisture tolerance. The molecular markers of the present invention are detected using 3% agarose gel, are stable, and have a simple method. They can be used to screen barley moisture tolerance at an early stage and have great application value in barley moisture tolerance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A genome-wide association analysis of moisture tolerance in barley;

[0030] Figure 2 The transcription level of HvERF62 under moisture stress was analyzed;

[0031] Figure 3 Identification of the moisture tolerance phenotype of the HvERF62 mutant; A: Gene sequence analysis of the HvERF62 mutant and the wild type HvERF62; B: Analysis of the moisture tolerance phenotype of the HvERF62 mutant and the wild type.

[0032] Figure 4Haplotype sequence analysis and developed InDel molecular markers for HvERF62. A: Haplotype analysis of HvERF62 in 334 barley varieties in natural populations; B: Analysis of differences in moisture tolerance among three haplotypes; C: Insertion / deletion sequence analysis of the amplified sequence of InDel molecular markers; D: Gel imaging of Hap1 / 2 and Hap3; E: Differences in moisture tolerance between two genotypes (whether the amino acid terminates or not). DETAILED DESCRIPTION

[0033] In order to illustrate the technical scheme and technical purpose of the present invention, the present invention is further introduced below in conjunction with the accompanying drawings and specific embodiments. The following examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, the modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0035] Example 1: Identification of candidate genes for moisture tolerance in barley

[0036] The present invention uses 334 two-rowed barley germplasm populations from different sources as materials, and the materials come from 14 countries or regions around the world. The seeds are placed on moist filter paper and germinated in a constant temperature incubator (25°C, dark) for 24 hours. After the seeds turn white, they are transplanted into potted plants. The pots are filled with special matrix nutrient soil for seedling cultivation. Four white seeds are evenly sown in each pot. When the seedlings grow to 3 leaves and 1 heart, they are treated with moisture damage, flooded to the base of the seedling stems, and normal water supply treatment is used as a control. When the leaves of some materials turn yellow and the groups show significant differences, the moisture damage treatment is stopped. The relative chlorophyll content of the leaves under control and moisture stress is determined using a SAPD instrument. The experiment was repeated 3 times.

[0037] The present invention performed full SLAF tag sequencing on the natural barley population based on the Illumina sequencing platform. After quality control, the data was strictly filtered according to the criteria of minimum allele frequency > 5% and deletion rate < 20%, and a total of 233,451 SNPs were obtained from the SLAF tag. The MLM linear mixed model of the R package "rMVP" was used for genome-wide association analysis, and the results were as follows: Figure 1As shown in the figure, a locus stably associated with moisture tolerance was identified on chromosome 4. Three moisture-tolerant varieties and three moisture-sensitive varieties were used as materials. After 60 h of moisture damage treatment, root tissues of the control and treated varieties were taken for qPCR analysis. The results showed that the expression level of HvERF62, a transcription factor encoding barley ethylene response, was high in the three moisture-tolerant materials and upregulated, but not in the three moisture-sensitive materials. Therefore, HvERF62 was identified as a candidate gene regulating barley moisture tolerance.

[0038] Example 2: HvERF62 gene cloning and sequence analysis

[0039] 2.1 Creation and phenotypic analysis of HvERF62 transgenic materials

[0040] Golden barley (GP) was used as the material, and the target site was designed according to the HvERF62 gene sequence. The primer sequences were SEQ ID NO.3 and SEQ ID NO.4. The CRISPR-Cas9 recombinant vector was constructed, and the plasmid was extracted using a plasmid miniprep kit (DC201, Norwexan) to transform Agrobacterium AGL1. The bacterial solution was aspirated with a 20ul pipette to infect the immature embryonic callus of golden barley. After infection, the callus with vigor, no bacterial contamination and a large amount of Agrobacterium growth was selected, and transferred to a medium containing hygromycin and cultured in a dark room at 28°C; the new medium was replaced every two weeks for a total of four weeks. During this period, calli with good growth status were selected for differentiation on the differentiation medium, and the surface of the culture dish was covered with white paper to create a weak light environment, and then cultured under 28°C light conditions; the differentiation medium was replaced every two weeks to remove the calli that were whitish, brown, and soft in texture. When the callus differentiated into seedlings and grew to 2-3 cm, the seedlings were transferred to the rooting medium and cultured under light. When the root system was established, the seedlings were transferred to the soil, and the seedling DNA was extracted. Then, the editing site was detected using the specific primers SEQ ID NO.5 and SEQ ID NO.6 containing the target site to analyze whether the editing was successful. Finally, three different homozygous mutant lines were obtained, namely, homozygous mutant lines with a deletion of 5 bp and an insertion of 1 bp, and the encoded proteins all had frameshift mutations ( Figure 3 ).

[0041] 2.2 Analysis of the moisture tolerance phenotype of the HvERF62 mutant

[0042] The seeds of golden barley and three HvERF62 homozygous mutant lines with complete and uniform plumpness were selected for germination, and the seeds with uniform germination were selected and sown in pots filled with mixed substrate. When they grew to the 2-leaf 1-heart stage, they were flooded. After 2 weeks, the materials were collected and the fresh weight and dry weight of the aboveground and underground parts were weighed to analyze the differences between the wild type and the edited mutants. The results showed that under the wet damage treatment, the root length, aboveground and underground biological weight of the three HvERF62 knockout mutants were significantly lower than those of the wild type ( Figure 3 ).

[0043] Example 3 HvERF62 haplotype analysis and related InDel marker development

[0044] The gene DNA is 1103 bp long, including one intron, and the CDS is 987 bp long, as shown in SEQ ID NO.1, encoding 328 amino acids, and the corresponding amino acid sequence is shown in SEQ ID NO.2. The protein has a molecular weight of 35.03 KDa, an isoelectric point of 5.64, and contains an AP2 / ERF functional domain located at amino acid positions 109-172 ( Figure 4 A). Analysis of the sequences of HvERF62 in 334 varieties revealed natural variation in HvERF62, with three main haplotypes. Among them, haplotype Hap3 had a frameshift mutation due to multiple base deletions, leading to premature termination of protein translation ( Figure 4 A). Under moisture stress, the chlorophyll content of haplotypes Hap1 and Hap2 was significantly higher than that of Hap3, while there was no significant difference in chlorophyll content between the controls ( Figure 4 B). Based on the base deletion difference, the InDel molecular marker HvERF62-InDel was developed. The sequences are shown in SEQ ID NO.7 and SEQ ID NO.8. The primers amplified products with a total length of 207 bp in haplotypes Hap1 and Hap2, and a total length of 180 bp in haplotype Hap3, with a difference of 27 bp ( Figure 4C). The primers are used to perform PCR amplification of the DNA fragment labeled with the InDel molecule of the sample to be tested, and a PCR amplification product is obtained, and the product sequence is shown in SEQ ID No.9 and SEQ ID No.10. The length of the PCR amplification product is analyzed, and the length of the nucleotide sequence shown in SEQ ID No.9 is 207 bp, and the length of the nucleotide sequence shown in SEQ ID No.10 is 180 bp. From the 5' end, the 39th to 42nd bases of the nucleotide sequence shown in SEQ ID NO.9 are CCCA, the 57th to 59th bases are GTC, the 99th to 116th bases are CTGCGGGCGGAGGAGGAC, and the 180th to 181st bases are TA, and the genotyping is Hap1 / 2 type, which is a moisture damage resistant material; while the nucleotide sequence shown in SEQ ID NO.10 has base deletions at the above nucleotide positions, and the genotyping is Hap3 type, which is a moisture damage sensitive material. If the length of the PCR amplification product is 180 bp, the sample to be tested is judged to be a moisture-sensitive material. If the length of the PCR amplification product is 207 bp, the sample to be tested is judged to be a moisture-resistant material. This marker is polymorphic in 3% agarose gel, and the difference is obvious ( Figure 4 D). This marker was used to type 334 varieties, and the chlorophyll content in the two groups was significantly different ( Figure 4 E), can be effectively used for the screening of barley varieties with moisture tolerance.

[0045] SEQ ID NO.1

[0046] ATGTGTGGCGGCGCCATCCTAGCGCAGCTGATCCCGCCGTCGGCGGGCCGTCCGTCGAAGCAGGCGGCAGCGGGCGGCCGGGCCCCGCCCACGAGCTCCAAGAAGGGCGGCGTGAGCAAGAGCCGCCACAGCAGCACCCCAGATGCCGACGACGACGTCTTCGAGGCCGCCTTCGAGGACTTCGATGACCACTTCGACCTGCGGGCGGAGGAGGACGGCGGCGACGACCATGTCGTCTTTGCATCCAAGCCTGCCTTCTCTCCACGTCCGGCCTACGACGGTGGCCGCGCGGCGCATGCGGCGAGCAGGAAGAAGCGCACCGGCCACCTCCATGGCATCCGGCAGCGGCCGTGGGGCAAGTGGGCGGCGGAGATCCGCGACCCGCACAAGGGCACCCGCGTCTGGCTCGGCACGTTCGACACGGCCGATGATGCCGCCCGGGCCTACGACGTCGCCGCCCGTCGCCTCCGTGGCAGCAAGGCCAAGGTCAACTTCCCCGACGCGGCCAGGACCGGGGCTCGCCCGCGCCGCGCCAGCCGTAGAACCGCGCAGAAACCGCAATGCCCCCCTGCGCGGACGACGGCGTACTCTGCCACCGCAGCAGCACGCGCACAGCCGGAGCAGGACGCTATGATGGTCAAACCCGAGCTGATGGAGTTTTTCAACGTGGACGCCATCGTCCACCTGACCACTGCCGTCGCCGCGCTACCGCCTGTCACGGCGAGCACCTTCGCCGACACGATGCCGAGGGTCGACGAGGACTCTTCTGTGGGGAGCGGCGGCGGCGCCATGCTGGGGTTCGCCGACGAGCTTGGGTTCGATCCGTTCATGATGTTCCAGCTACCCTGCTCGGACATGTACGAATCCGCCGACAGCATCTTCGCCGGAGACGCTGTCATCCCGGATGCCCTCAGCGTGGACAGTGGCATGGACGCCGTCAGCCTCTGGAGCTTCGACGAGTTCCCCATGGACAGCGCCATTTTCTGA。

[0047] SEQ ID NO.2

[0048] MCGGAILAQLIPPSAGRPSKQAAAGGRAPPTSSKKGGVSKSRHSSTPDADDDVFEAAFEDFDDHFDLRAEEDGGDDHVVFASKPAFSPRPAYDGGRAAHAASRKKRTGHLHGIRQRPWGKWAAEIRDPHKGTRVWLGTFDTADDAARAYDVAARRLRGSKAKVNFPDAARTGARPRRASRRTAQKPQCPPARTTAYSATAAARAQPEQDAMMVKPELMEFFNVDAIVHLTTAVAALPPVTASTFADTMPRVDEDSSVGSGGGAMLGFADELGFDPFMMFQLPCSDMYESADSIFAGDAVIPDALSVDSGMDAVSLWSFDEFPMDSAIF。

[0049] SEQ ID NO.3: 5'-cttgCCTCCATGGCATCCGGCAG-3'。

[0050] SEQ ID NO.4: 5'-aaacCTGCCGGATGCCATGGAGG-3'。

[0051] SEQ ID No.5: 5'-ATGTGTGGCGGCGCCATCCTAG-3'。

[0052] SEQ ID No.6: 5'-GGCATCATCGGCCGTGTCGAAC-3'。

[0053] SEQ ID No.7: 5’-AGAAGGGCGGCGTGAGCAAGAG-3’。

[0054] SEQ ID No.8: 5’-GCACACTCAGATGAAACTATGAGAG-3’。

[0055] SEQ ID No.9:

[0056] AGAAGGGCGGCGTGAGCAAGAGCCGCCACAGCAGCACCCCAGATGCCGACGACGACGTCTTCGAGGCCGCCTTCGAGGACTTCGATGACCACTTCGACCTGCGGGCGGAGGAGGACGGCGGCGACGACCATGTCGTCTTTGCATCCAAGCCTGCCTTCTCTCCACGTCCGGGTAGGCTATAACTCTCATAGTTTCATCTGAGTGTGC。

[0057] SEQ ID No.10:

[0058] AGAAGGGCGGCGTGAGCAAGAGGCGCCACACCAGCACTATGCCGACGACGACTTCGAGGCCGCCTTCGAGGACTTCGATGACGACCTCGACGGCGGCGGCGACCATGTCGTTTTTGCATCCAAGCTTGCGCTCTCTCCGGGTCCAGGTAGGGTAACTCTCATAGTTTCATCTGAGTGTGC。

Claims

1. A gene HvERF62 regulating barley moisture tolerance, characterized in that: The nucleotide sequence of the barley moisture tolerance gene HvERF62 is shown in SEQ ID NO.

1.

2. A protein HvERF62 that regulates barley moisture tolerance, characterized in that: Encoded by the gene of claim 1, the sequence of the barley moisture tolerance protein HvERF62 is shown in SEQ ID NO.

2.

3. Use of the gene according to claim 1 or the protein according to claim 2 in regulating moisture tolerance of barley.

4. A method for regulating moisture tolerance of barley, characterized in that: The method comprises changing the content and / or activity of the protein according to claim 2 in the target plant to obtain a HvERF62 mutant plant.

5. The method according to claim 4, characterized in that Using CRISPR / Cas9 gene editing technology, a recombinant plasmid pCas9-HvERF62 expressing the Cas9 protein gene and the sgRNA gene was constructed, and HvERF62 mutant transgenic plants were cultured.

6. The method according to claim 5, characterized in that The target site primer sequences of sgRNA are shown in SEQ ID No.3 and SEQ ID No.

4.

7. The method according to claim 5, characterized in that The primer sequences for detecting transgenic plants are shown in SEQ ID No.5 and SEQ ID No.

6.

8. An InDel molecular marker for regulating moisture tolerance of barley, characterized in that: The InDel molecular marker primers for regulating barley moisture tolerance are shown in SEQ ID NOs. 7 and 8.

9. The InDel molecular marker for regulating barley moisture tolerance according to claim 8, characterized in that: The sequences of the PCR products amplified by the InDel molecular marker in the natural population are shown in SEQ ID No.9 and SEQ ID No.10; the nucleotide sequence shown in SEQ ID No.9 is 207 bp in length, and the nucleotide sequence shown in SEQ ID No.10 is 180 bp in length; from the 5' end, the 39th to 42nd bases of the SEQ ID NO.9 sequence are CCCA, the 57th to 59th bases are GTC, the 99th to 116th bases are CTGCGGGCGGAGGAGGAC, and the 180th to 181st bases are TA, and the genotyping is Hap1 / 2 type, which is a moisture damage-resistant material; while the nucleotide sequence shown in SEQ ID NO.10 has base deletions at the above nucleotide positions, and the genotyping is Hap3 type, which is a moisture damage-sensitive material.

10. Use of the InDel molecular marker for regulating moisture tolerance of barley according to claim 8 or 9 in the screening of moisture-tolerant barley varieties and molecular marker-assisted selection breeding.

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