Gene hv Erf62 for regulating wet tolerance of barley and application thereof

Abstract

The present application belongs to the field of molecular genetics, and particularly relates to an ethylene response transcription factor HvERF62 for regulating the moisture tolerance of barley and application thereof in molecular marker assisted selection breeding. A site significantly affecting the moisture tolerance of barley is identified through whole genome association analysis, and a candidate gene is predicted, and further, the moisture tolerance function of the candidate gene HvERF62 is verified by using the CRISPR-Cas9 gene editing method. The function of HvERF62 for regulating the moisture tolerance of barley is determined by using the gene editing technology, and an InDel molecular marker for detecting the moisture tolerance of barley is developed. The molecular marker of the present application is detected by using 3% agarose gel, is stable, simple in method, and can be used for screening the moisture tolerance of barley at an early stage, and has a strong application value in the breeding of the moisture tolerance of barley.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular genetics, and particularly relates to a gene HvERF62 for regulating the wet tolerance of barley and an application thereof. BACKGROUND

[0002] Barley is a crop sensitive to wet damage. Under wet stress, too much water hinders the transmission of oxygen in the air to the plant, causing the root system to be in a low-oxygen or even anaerobic state, directly affecting the normal physiological activities of the root system, and thus leading to reduced yield, or even no harvest. The influence of wet stress on plant phenotypic traits mainly manifests in wilting, drooping, yellowing, and even rotting of leaves, inhibition of new leaf formation, reduction of total leaf area, blackening and rotting of roots, induction of adventitious roots, and formation of rhizosphere aeration tissues. If wet damage occurs during the germination or seedling establishment period of barley, it will greatly reduce the germination rate of seeds and the survival rate of seedlings, and if it occurs during the middle and late growth periods of barley, it will greatly affect the growth of barley and reduce its yield and quality.

[0003] Numerous studies have shown that the formation of aeration tissues and adventitious roots after flooding is related to the accumulation of ethylene, and ethylene response factors (ERFs) play an important role. Rice can regulate the expression of ERFs genes through ethylene signaling to promote or inhibit internode elongation to adapt to different types of wet stress. Overexpression of ERF genes in crops such as Arabidopsis, wheat, and rape can improve wet tolerance. However, there is no report on the involvement of ERF genes in the wet tolerance of barley through transgenic means.

[0004] Research on plant wet tolerance is crucial for improving crop wet tolerance to cope with increasingly severe extreme weather and large-scale production management. In recent years, breakthroughs have been made in the research on ordinary wild rice and Arabidopsis as model plants, making us have a deeper understanding of the response mechanism to wet stress. However, few wet tolerance genes have been cloned in barley. Therefore, we urgently need to explore key genes related to the wet tolerance of barley to provide gene resources and molecular markers for barley wet tolerance breeding. SUMMARY

[0005] The application aims to provide a gene HvERF62 capable of regulating the wet tolerance of barley and an excellent haplotype thereof, and develop an InDel molecular marker based on the excellent haplotype, so as to provide a gene resource and a molecular marker for the identification of wet tolerance materials of barley and the breeding of varieties.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the application is:

[0007] A gene HvERF62 capable of regulating the wet tolerance of barley, wherein the nucleotide sequence of the wet tolerance gene HvERF62 of the barley is shown as SEQ ID NO. 1, and the CDS of the gene has a full length of 987 bp.

[0008] Further, the amino acid sequence of the ethylene response transcription factor HvERF62 of the barley is shown as SEQ ID NO. 2. The protein is composed of 328 amino acid residues, and the corresponding amino acid sequence is shown as SEQ ID NO. 2. The protein has a molecular weight of 35.03 KDa, an isoelectric point of 5.64, and a functional domain prediction analysis shows that it contains one AP2 / ERF functional domain.

[0009] The application further provides the application of the above-mentioned gene or protein in regulating the wet tolerance of barley.

[0010] The application further provides a method for regulating the wet tolerance of barley, which comprises regulating the content and / or activity of the above-mentioned protein in a plant of interest, so as to obtain a mutant plant.

[0011] Further, the CRISPR / Cas9 gene editing technology is used to construct a recombinant plasmid pCas9-HvERF62 expressing the genes of Cas9 protein and sgRNA, and a transgenic plant is obtained through culture.

[0012] Further, the primer sequence of the target site of sgRNA is shown as SEQ ID No. 3 and SEQ ID No. 4.

[0013] Further, the primer sequence of the transgenic plant is shown as SEQ ID No. 5 and SEQ ID No. 6.

[0014] The barley ethylene response transcription factor HvERF62 has natural variations in barley, including Hap1, Hap2 and Hap3 haplotypes.

[0015] Further, the correlation between the HvERF62 haplotype and the chlorophyll content under wet stress is analyzed, and an excellent HvERF62 haplotype is identified, which can be applied to improve the wet tolerance of barley.

[0016] Further, according to the sequence differences between the haplotypes, an InDel molecular marker is developed for identifying and molecular marker assisted selection breeding of barley wet tolerance varieties. The InDel molecular marker, the primer or the detection kit is applied to detect or identify the wet tolerance breeding of barley.

[0017] The application also provides an InDel molecular marker for regulating the wet tolerance of barley, and the sequence of the primer of the InDel molecular marker for regulating the wet tolerance of barley is shown in SEQ ID No. 7 and SEQ ID No. 8.

[0018] Further, the sequence of the PCR product amplified by the InDel molecular marker SEQ ID No. 7 and SEQ ID No. 8 is shown in SEQ ID No. 9 and SEQ ID No. 10; the bases at positions 39-42, 57-59, 99-116, 180-181 of the sequence of SEQ ID No. 9 from the 5' end are CCC A, GTC, CTGCGGGCGGAGGAGGAC and TA, respectively, and the genotyping is Hap1 / 2 type, which is a wet tolerance material; and the nucleotide sequence shown in SEQ ID No. 10 lacks the bases at the above nucleotide positions, and the genotyping is Hap3 type, which is a wet sensitive material.

[0019] The application also provides the application of the above-mentioned InDel molecular marker for regulating the wet tolerance of barley in screening of barley wet tolerance varieties and molecular marker assisted selection breeding.

[0020] Further, the reaction procedure of the PCR amplification is 94℃ 5min; 94℃ 30sec, 55℃ 30sec, 72℃ 1.5min, 32 cycles; and 72℃ 10min.

[0021] Further, the reaction system of the PCR amplification is 2×PCR MasterMix 5μL, forward primer 0.5μL, reverse primer 0.5μL, genomic DNA 0.5μL, and sterile deionized water is supplemented to 10μL.

[0022] Further, the length of the PCR amplification product is used for genotype detection and analysis of the sample to be tested using 3% agarose gel. Advantages

[0023] The application provides a new gene HvERF62 capable of regulating the moisture tolerance of barley, and the gene HvERF62 positively regulates the moisture tolerance of barley, thereby providing a gene resource for breeding of barley with moisture tolerance.

[0024] HvERF62 mainly exists in three haplotypes in 334 barley varieties, and the haplotype 3 significantly reduces the moisture tolerance due to early termination of amino acid coding caused by base deletion. The application screens an excellent haplotype Hap1 with moisture tolerance in a natural population of barley, and the haplotype Hap1 can be applied to genetic improvement of the moisture tolerance of barley.

[0025] The application provides an InDel molecular marker, and provides a tool for screening of barley varieties with moisture tolerance and molecular marker assisted selection breeding.

[0026] The application determines the function of HvERF62 in regulating the moisture tolerance of barley through gene editing technology, and develops an InDel molecular marker for detection of the moisture tolerance of barley, the molecular marker of the application is detected using 3% agarose gel, is stable in performance, is simple in method, can be used for early screening of the moisture tolerance of barley, and has strong application value in breeding of barley with moisture tolerance. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Whole genome association analysis of the moisture tolerance of barley;

[0028] Figure 2 Transcription level analysis of HvERF62 under wet stress;

[0029] Figure 3 Phenotype identification of the moisture tolerance of HvERF62 mutants; A: gene sequence analysis of HvERF62 mutants and wild type HvERF62; B: moisture tolerance phenotype analysis of the HvERF62 mutants and the wild type.

[0030] Figure 4 Haplotypes sequence analysis of HvERF62 and development of an InDel molecular marker; A: haplotype analysis of HvERF62 in 334 barley varieties in a natural population; B: moisture tolerance difference analysis of three haplotypes; C: insertion / deletion sequence analysis of the amplification sequence of the InDel molecular marker; D: gel imaging diagram of Hap1 / 2 and Hap3; E: moisture tolerance difference of two genotypes (whether the amino acid terminates). DETAILED DESCRIPTION

[0031] In order to clarify the technical solutions and technical purposes of the present application, the present application will be further described below in combination with the drawings and specific embodiments. The following examples are only used to illustrate the present application and are not used to limit the application range. Any modification or replacement of the method, step or condition of the present application without departing from the spirit and essence of the present application shall fall within the scope of the present application.

[0032] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials available from commercial channels unless otherwise specified.

[0033] Example 1: Identification of a candidate gene for barley moisture tolerance

[0034] The present application uses 334 different sources of two-edged barley germplasm population as materials, which are from 14 countries or regions around the world. The seeds are placed on wet filter paper and germinated in a constant temperature incubator (25℃, dark) for 24 h. After the seeds are white, they are transplanted into pots filled with special substrate nutrient soil. Each pot is uniformly sown with 4 white seeds. When the seedlings grow to 3 leaves 1 heart, they are subjected to wet damage treatment, which is flooded to the stem base of the seedlings. The normal water supply treatment is used as a control. When the leaves of some materials turn yellow and the population shows significant differences, the wet damage treatment is stopped. The relative content of chlorophyll in the leaves of the control and wet stress is determined by SAPD instrument. The experiment is set up with 3 replicates.

[0035] Based on the Illumina sequencing platform, the above barley natural population is subjected to SLAF tag whole sequencing. After data quality control, 233,451 SNPs are obtained from the SLAF tag after strict filtering according to the standards of minimum allele frequency > 5% and deletion rate < 20%. The whole genome association analysis is carried out by using the MLM linear mixed model of R package "rMVP", and the results are shown in Figure 1 As shown in the figure, a site stably associated with moisture tolerance is identified on chromosome 4. After 60 h of wet damage treatment, the root tissues of the control and the treatment are taken, and qPCR analysis is carried out. The results show that the expression of barley ethylene response transcription factor HvERF62 is higher in 3 moisture tolerant materials, and is up-regulated, while it is not expressed in 3 moisture sensitive materials. Therefore, HvERF62 is locked as a candidate gene for regulating barley moisture tolerance.

[0036] Example 2: Cloning and sequence analysis of HvERF62 gene

[0037] 2.1 Creation of HvERF62 transgenic materials and phenotype analysis

[0038] Golden barley (GP) as the material, according to the sequence of HvERF62 gene, the target site is designed, the primer sequence is SEQ ID NO. 3 and SEQ ID NO. 4. Construct CRISPR-Cas9 recombinant vector, use plasmid small amount extraction kit (DC201, novozyme) to extract plasmid, transform agrobacterium AGL1. Use 20ul pipette to suck bacteria liquid to infect golden barley young embryo callus, co-culture for 3 days after infection, then pick up the callus with vigorous vitality, no contamination and a large number of agrobacterium growth, transfer to the medium containing hygromycin, cultivate at 28℃ in dark room; Replace new medium every two weeks, a total of four weeks. During this period, the callus with good growth state is selected for differentiation on differentiation medium, white paper is used to cover the surface of culture dish to create weak light environment, then cultivate under 28℃ light condition; Replace differentiation medium every two weeks, remove the callus with whitish, brown and soft texture. When the callus is differentiated into seedlings and grows to 2-3cm, the seedlings are transferred to rooting medium and cultured under light. When the root system is established, the seedling DNA is extracted, and then the specific primers containing target site SEQ ID NO. 5 and SEQ ID NO. 6 are used to detect the editing site to analyze whether the editing is successful. Finally, three different homozygous mutant systems are obtained, which are 5bp deletion and 1bp insertion homozygous mutant lines, and the encoded proteins all have frame shift mutation Figure 3 ).

[0039] 2.2 HvERF62 mutant wet tolerance phenotype analysis

[0040] Select complete and consistent golden barley and three HvERF62 homozygous mutant system seeds for germination, select uniform germination seeds, and sow in pots with mixed substrate. When it grows to 2 leaf 1 heart stage, it is subjected to flooding treatment, and after 2 weeks, the aboveground and underground fresh and dry weights of the materials are collected and weighed to analyze the differences between wild type and edited mutants. The results show that under wet damage treatment, the root length, aboveground and underground biomass of three HvERF62 knockout mutants are extremely significantly lower than those of wild type Figure 3 ).

[0041] Example 3 HvERF62 haplotype analysis and related InDel marker development

[0042] The DNA full length of the gene is 1103 bp, containing an intron, the CDS full length is 987 bp, 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 molecular weight is 35.03 KDa, and the isoelectric point is 5.64. It contains an AP2 / ERF functional domain, which is located at amino acid 109-172 Figure 4A). The sequence of HvERF62 in 334 varieties was analyzed, and natural variation of HvERF62 was found, mainly including three haplotypes. The haplotype Hap3 was subjected to frame mutation due to multiple base deletions and caused premature termination of protein translation Figure 4 A). Under the stress of wet damage, the chlorophyll content of haplotype Hap1 and Hap2 was significantly higher than that of Hap3, and there was no significant difference in chlorophyll content between the control Figure 4 B). An InDel molecular marker HvERF62-InDel was developed according to the difference in base deletion, and the sequence is shown as SEQ ID NO. 7 and SEQ ID NO. 8. The primer amplification product is 207 bp in length in haplotype Hap1 and Hap2, and the amplification product is 180 bp in length in haplotype Hap3, with a difference of 27 bp Figure 4 C). The InDel molecular marker DNA fragment of the test sample was amplified by PCR using the primer, and the PCR amplification product was obtained, and the product sequence is shown as SEQ ID No. 9 and SEQ ID No. 10. The length of the PCR amplification product was analyzed, and the length of the nucleotide sequence shown as SEQ ID No. 9 was 207 bp, and the length of the nucleotide sequence shown as SEQ ID No. 10 was 180 bp. The 39-42 bases of the nucleotide sequence shown as SEQ ID No. 9 from 5' end were CCC A, the 57-59 bases were GTC, the 99-116 bases were CTGCGGGCGGAGGAGGAC, the 180-181 bases were TA, and the genotyping was Hap1 / 2, which was a wet damage tolerant material. The base deletion of the nucleotide sequence shown as SEQ ID No. 10 at the above nucleotide position was Hap3 type, which was a wet damage sensitive material. If the length of the PCR amplification product is 180 bp, the test sample is a wet damage sensitive material, and if the length of the PCR amplification product is 207 bp, the test sample is a wet damage tolerant material. The marker has polymorphism in 3% agarose gel, and the difference is obvious Figure 4 D). The marker was used to genotype 334 varieties, and the chlorophyll content was significantly different in 2 groups Figure 4 E), which can be effectively used for screening of wet tolerance varieties of barley.

[0043] SEQ ID NO. 1

[0044] ATGTGTGGCGGCGCCATCCTAGCGCAGCTGATCCCGCCGTCGGCGGGCCGTCCGTCGAAGCAGGCGGCAGCGGGCGGCCGGGCCCCGCCCACGAGCTCCAAGAAGGGCGGCGTGAGCAAGAGCCGCCACAGCAGCACCCCAGATGCCGACGACGACGTCTTCGAGGCCGCCTTCGAGGACTTCGATGACCACTTCGACCTGCGGGCGGAGGAGGACGGCGGCGACGACCATGTCGTCTTTGCATCCAAGCCTGCCTTCTCTCCACGTCCGGCCTACGACGGTGGCCGCGCGGCGCATGCGGCGAGCAGGAAGAAGCGCACCGGCCACCTCCATGGCATCCGGCAGCGGCCGTGGGGCAAGTGGGCGGCGGAGATCCGCGACCCGCACAAGGGCACCCGCGTCTGGCTCGGCACGTTCGACACGGCCGATGATGCCGCCCGGGCCTACGACGTCGCCGCCCGTCGCCTCCGTGGCAGCAAGGCCAAGGTCAACTTCCCCGACGCGGCCAGGACCGGGGCTCGCCCGCGCCGCGCCAGCCGTAGAACCGCGCAGAAACCGCAATGCCCCCCTGCGCGGACGACGGCGTACTCTGCCACCGCAGCAGCACGCGCACAGCCGGAGCAGGACGCTATGATGGTCAAACCCGAGCTGATGGAGTTTTTCAACGTGGACGCCATCGTCCACCTGACCACTGCCGTCGCCGCGCTACCGCCTGTCACGGCGAGCACCTTCGCCGACACGATGCCGAGGGTCGACGAGGACTCTTCTGTGGGGAGCGGCGGCGGCGCCATGCTGGGGTTCGCCGACGAGCTTGGGTTCGATCCGTTCATGATGTTCCAGCTACCCTGCTCGGACATGTACGAATCCGCCGACAGCATCTTCGCCGGAGACGCTGTCATCCCGGATGCCCTCAGCGTGGACAGTGGCATGGACGCCGTCAGCCTCTGGAGCTTCGACGAGTTCCCCATGGACAGCGCCATTTTCTGA.

[0045] SEQ ID NO. 2

[0046] MCGGAILAQLIPPSAGRPSKQAAAGGRAPPTSSKKGGVSKSRHSSTPDADDDVFEAAFEDFDDHFDLRAEEDGGDDHVVFASKPAFSPRPAYDGGRAAHAASRKKRTGHLHGIRQRPWGKWAAEIRDPHKGTRVWLGTFDTADDAARAYDVAARRLRGSKAKVNFPDAARTGARPRRASRRTAQKPQCPPARTTAYSATAAARAQPEQDAMMVKPELMEFFNVDAIVHLTTAVAALPPVTASTFADTMPRVDEDSSVGSGGGAMLGFADELGFDPFMMFQLPCSDMYESADSIFAGDAVIPDALSVDSGMDAVSLWSFDEFPMDSAIF.

[0047] SEQ ID NO. 3: 5'-cttgCCTCCATGGCATCCGGCAG-3'.

[0048] SEQ ID NO. 4: 5'-aaacCTGCCGGATGCCATGGAGG-3'.

[0049] SEQ ID No. 5: 5'-ATGTGTGGCGGCGCCATCCTAG-3'.

[0050] SEQ ID No. 6: 5'-GGCATCATCGGCCGTGTCGAAC-3'.

[0051] SEQ ID No. 7: 5'-AGAAGGGCGGCGTGAGCAAGAG-3'.

[0052] SEQ ID No. 8: 5'-GCACACTCAGATGAAACTATGAGAG-3'.

[0053] SEQ ID No. 9:

[0054] AGAAGGGCGGCGTGAGCAAGAGCCGCCACAGCAGCACCCCAGATGCCGACGACGACGTCTTCGAGGCCGCCTTCGAGGACTTCGATGACCACTTCGACCTGCGGGCGGAGGAGGACGGCGGCGACGACCATGTCGTCTTTGCATCCAAGCCTGCCTTCTCTCCACGTCCGGGTAGGCTATAACTCTCATAGTTTCATCTGAGTGTGC.

[0055] SEQ ID No. 10:

[0056] AGAAGGGCGGCGTGAGCAAGAGGCGCCACACCAGCACTATGCCGACGACGACTTCGAGGCCGCCTTCGAGGACTTCGATGACGACCTCGACGGCGGCGGCGACCATGTCGTTTTTGCATCCAAGCTTGCGCTCTCTCCGGGTCCAGGTAGGGTAACTCTCATAGTTTCATCTGAGTGTGC.

Claims

1. The application of InDel molecular marker for regulating the moisture tolerance of barley in the screening of barley moisture tolerance varieties and the molecular marker assisted selection breeding of barley moisture tolerance varieties, characterized in that, The InDel molecular marker for regulating the wet tolerance of barley is obtained by PCR amplification with primers shown as SEQ ID NO. 7 and 8; if the length of the PCR amplification product is 180 bp, the sample to be tested is judged as a wet damage sensitive material, and if the length of the PCR amplification product is 207 bp, the sample to be tested is judged as a wet damage resistant material.

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