Application of excellent natural variation of GmPOD123 gene in regulation of drought resistance in soybean germination period

Abstract

The present invention discloses GmPOD123 The invention discloses an application of excellent natural variation of genes in drought resistance regulation of soybean during germination period, and relates to the field of biotechnology. GmPOD123 Gene association analysis of drought resistance phenotypes during germination period in 400 soybean germplasm resource populations was conducted to explore and verify GmPOD123 The gene has excellent natural variation that is significantly associated with drought resistance in soybean germination. Further research results show that overexpression GmPOD123 HapG The transgenic strains of the peroxidase gene (HapG, Chr17:14532734) can promote soybean germination under drought stress, which proves that the gene can improve the drought resistance of soybean during the germination period. GmPOD123 This not only enriches the candidate gene resources for improving soybean drought resistance during the germination period, but also provides candidate molecular markers for breeding new drought-resistant soybean germplasm.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology and specifically relates to GmPOD123 Application of genetically superior natural variation in regulating drought resistance in soybean during germination period. Background Art

[0002] Soybean ( Glycinemax ) is one of my country's important food crops, but my country's soybean imports are as high as 100.327 million tons, while the production is only 20 million tons, with a serious shortage of supply and demand. Drought is one of the main natural disasters that affect the germination of soybean seeds, seriously affecting the changes in physiological and biochemical indicators such as soluble sugar, fat, and proline in seeds, inhibiting the emergence rate of soybeans, and ultimately leading to a reduction in soybean production.

[0003] POD is a plant-specific peroxidase that can remove excess ROS in plants. Therefore, through the catalytic cycle of peroxidase, POD can degrade ROS accumulated in plants, maintain ROS at a relatively stable level, and thus increase the stress resistance of plants. In recent years, the peroxidase family has been widely studied in many species, such as Arabidopsis, corn, potatoes, carrots, and wheat, but the members and functions of the POD gene family in soybeans are still unclear, especially GmPOD123 The relationship between genes and drought resistance in soybean during germination is still unclear. Summary of the invention

[0004] In view of the above-mentioned defects of the prior art, the present invention proposes GmPOD123 The application of genetically superior natural variation in the regulation of soybean drought resistance during the germination period is to solve the problems raised by the above-mentioned background technology.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides an application of a SNP site associated with drought resistance of soybean during germination period in improving drought resistance of plants during germination period, wherein the SNP site is located at GmPOD123 At position 145 of the gene, there is a natural A / G variation at this site;

[0007] Said GmPOD123 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0008] Preferably, the drought resistance of soybean during germination is manifested as follows: the germination rate and hypocotyl length of soybean germplasm containing haplotype I under drought stress are significantly higher than those of soybean germplasm containing haplotype II;

[0009] The haplotype I is: soybean germplasm of HapG genotype;

[0010] The haplotype II is soybean germplasm of HapA genotype.

[0011] Preferably, by overexpression GmPOD123 The gene further improves the drought resistance of plants during the germination period;

[0012] The plant is soybean.

[0013] Preferably, the GmPOD123 The gene cloning method includes the following steps:

[0014] (1) Extract RNA from soybean plants and obtain cDNA by reverse transcription;

[0015] (2) using the cDNA as a template and the primer pair to perform PCR amplification to obtain the gene sequence shown in SEQ ID NO: 1;

[0016] The primer pair includes GmPOD123-F and GmPOD123-R;

[0017] The nucleotide sequence of GmPOD123-F is shown in SEQ ID NO: 2;

[0018] The nucleotide sequence of GmPOD123-R is shown in SEQ ID NO: 3;

[0019] The variety of the soybean plant is: Changnong 26.

[0020] Preferably, the conditions for PCR amplification in step (2) are: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, for a total of 35 cycles, and post-extension at 72°C for 8 min.

[0021] The present invention provides a method for improving drought resistance of soybeans in the germination period, comprising the following steps:

[0022] S1. Connect the gene sequence shown in SEQ ID NO: 1 to the starting vector to obtain an overexpression vector;

[0023] S2, transferring the overexpression vector obtained in step S1 into the starting strain to obtain a recombinant engineered bacterium;

[0024] S3, infect soybean with the recombinant engineering bacteria obtained in step S2 to obtain overexpression GmPOD123 of genetically modified soybeans.

[0025] Preferably, the starting vector in step S1 is a pCAMBIA3301-Flag vector.

[0026] Preferably, the starting strain in step S2 is a prokaryotic microorganism or a eukaryotic microorganism.

[0027] Preferably, the starting strain is EHA105 Agrobacterium.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention is through GmPOD123 Gene association analysis of drought resistance phenotypes during germination period in 400 soybean germplasm resource populations was conducted to explore and verify GmPOD123 The gene has excellent natural variation that is significantly associated with drought resistance in soybean germination. Further research results show that overexpression GmPOD123 HapG The transgenic strains of the gene can promote soybean germination under drought stress, which proves that the gene can improve the drought resistance of soybean during the germination period. GmPOD123 This not only enriches the candidate gene resources for improving soybean drought resistance during the germination period, but also provides candidate molecular markers for breeding new drought-resistant soybean germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0031] Figure 1 Soybean provided by the present invention GmPOD123 Population analysis of significant SNP sites of genes;

[0032] Figure 2 Soybean provided by the present invention GmPOD123 Haplotype identification of genes;

[0033] Figure 3 Soybean provided by the present invention GmPOD123 PCR amplification diagram of the full-length cDNA fragment of the gene;

[0034] Figure 4 The invention provides GmPOD123 Plant overexpression vector map of genes;

[0035] Figure 5 The overexpression provided by the present invention GmPOD123 Identification of genetically modified soybeans;

[0036] Figure 6 The overexpression provided by the present invention GmPOD123 Identification of the expression level of transgenic soybeans;

[0037] Figure 7 Overexpression under drought stress provided by the present invention GmPOD123 Phenotypic identification of transgenic soybeans;

[0038] Figure 8Overexpression under drought stress provided by the present invention GmPOD123 Genetic analysis of transgenic soybeans. DETAILED DESCRIPTION

[0039] Unless otherwise specified, the reagents and methods used in the present invention are conventional reagents and methods.

[0040] Example 1 Soybean GmPOD123 Haplotype analysis of genes

[0041] In order to screen out the key genes for drought resistance in soybean germination, this project used 400 core soybean germplasms as research materials, combined with population germination period phenotypic data and whole genome association analysis, and based on haplotype analysis methods, found GmPOD123 The gene has excellent natural variation in the exon region, especially at Chr17:14532734, where there is a significant SNP variation (A / G), which leads to a non-synonymous amino acid mutation (threonine / alanine). The results are shown in Figure 1 .

[0042] To further evaluate the relationship between the two different haplotypes and drought resistance traits of soybean during germination, this study used the relative seed germination rate of soybean on the eighth day as an indicator and used the generalized linear model (GLM) in Tassel 5 software to analyze the relationship between the two different haplotypes and drought resistance traits of soybean during germination. GmPOD123 The gene haplotype was further identified. The results showed that GmPOD123 HapG The drought resistance level of soybean in the germination period was significantly higher than GmPOD123 HapA The results of the study showed that the Chr17:14532734 locus may have excellent allele variation related to drought resistance during the soybean germination period.

[0043] Example 2 GmPOD123 Haplotype identification of genes

[0044] Drought-resistant soybean varieties: Changnong 26, Beidou 36, Dongqing 25 and Huaidou 16; sensitive varieties: Haoyu 1, Heihe 49, Huajiang 5 and Jiadou 68 were selected, and total RNA was extracted from the seeds of the above varieties (Trizol extraction) and reverse transcribed into cDNA. The reverse transcription system is shown in Table 1.

[0045] Table 1 Reverse transcription system

[0046]

[0047] The conditions for reverse transcription are: the first step reaction conditions are: incubate at 42℃ for 2 min or at room temperature for 5 min; the second step reaction conditions are: 37℃ for 15 min, 85℃ for 5 s, 4℃. Storage: The cDNA obtained by reverse transcription was stored in a -20℃ refrigerator.

[0048] The primers GmPOD123-F and GmPOD123-R were used to GmPOD123 The gene was cloned, wherein the nucleotide sequence of GmPOD123-F was: 5'-ATGGGTCGGCTTAATCTCC-3' (SEQ ID No. SEQ ID NO: 2); the nucleotide sequence of GmPOD123-R was: 5'-TTAAAGAACAGAACAATCGGTACG-3' (SEQ ID NO: 3); the PCR reaction system was shown in Table 2; the PCR reaction program was: 95°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, a total of 35 cycles, and 72°C post-extension for 8 min.

[0049] Table 2 GmPOD123 Gene PCR system

[0050]

[0051] The PCR products amplified from samples Changnong 26, Beidou 36, Dongqing 25, Huaidou 16 and samples Haoyu 1, Heihe 49, Huajiang 5, Jiadou 68 were sequenced, and the gene sequences were compared with soybean Glycine max Wm82.a2.v1 as the reference genome. The results are shown in Figure 2 (exist Figure 2 The top row of sequences is the sequence of the reference genome).

[0052] Depend on Figure 2 It can be seen that GmPOD123 There is a SNP (A / G) variation in the gene.

[0053] Example 3 Soybean GmPOD123 Gene cloning

[0054] Using the cDNA of the day 0 seeds of the extremely drought-resistant soybean variety Changnong 26 as a template, primers GmPOD123-F (SEQ ID NO: 2) and GmPOD123-R (SEQ ID NO: 3) were used to generate the 5′-mercaptoethanol residues of the soybean seed. GmPOD123 The gene was cloned; the PCR reaction system is shown in Table 2; the PCR reaction program was: 95°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, a total of 35 cycles, and 72°C post-extension for 8 min.

[0055] The obtained PCR products were detected by agarose gel electrophoresis (for specific detection results, see Figure 3), and then connected to the cloning vector pEASY-T1 to generate pEASY-GmPOD123, and then sequenced. The sequencing result is shown in SEQ ID NO: 1.Specifically: 5'-ATGGGTCGGCTTAATCTCCTACTTGTGTGGTCACTCTCATTAACACTGTCCCTTTATTATCTACACCCAATCTCTGCTCAGCTCAGTCCAAACCACTATGCTAAAACATGCCCCAATCTTGAAAGCATCGTTAGACAGGCCGTTGCCAAGAAGTTCCAACAAACCTTTGTCACCGTCCCGGCCACCCTTCGTCTCTTCTTTCATGATTGCTTTGTGCAGGGTTGTGATGCTTCTGTTTTGATTGCATCCACCGGAAACAACCAAGCAGAGAAGGATCACCCAGACAACCTGTCTTTGGCGGGTGATGGGTTTGACACAGTGATCAAAGCAAAGGCTGCAGTAGATGCCATCCCTCAGTGCAGAAACAAAGTCTCATGTGCTGATATTCTGGCCTTGGCCACCCGTGATGTTATTGCACTGTCTGGTGGACCTTCATACACGGTTGAATTGGGAAGATTTGATGGTTTAGTTTCAAGAACTTCGGATGTGAATGGGAGGCTTCCACAGCCAACGAACAATTTGAACCAATTGAATTCACTGTTTGCGGCAAACGGCCTCACCCAAACTGACATGATTGCTCTCTCAGGTGCGCACACCCTTGGATTCTCCCACTGCAGCAAGTTCGCCAGTAGAATATACAGTACCCCAGTGGACCCAACACTGAACAAGCAATACGTAGCCCAATTGCAACAAATGTGCCCAAGGAACGTGGATCCTAGGATTGCCATCAACATGGACCCAACCACTCCTCGTAAATTCGACAACGTTTATTACCAAAACCTTCAACAAGGAAAGGGCCTCTTTACCTCGGACCAAATCCTCTTCACGGACCCAAGGTCCAGAAACACAGTTAATTCCTTCGCTTCTAGTAGCAACGTTTTCAACTCCAACTTTGTGGCTGCTATGACCAAATTGGGTCGTGTTGGAGTTAAGACTGCGAGAAATGGGAAAATTCGTACCGATTGTTCTGTTCTTTAA-3'.

[0056] The protein encoded by the sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 4, and the amino acid sequence of the protein is specifically: MGRLNLLLVWSLSLTLSLYYLHPISAQLSPNHYAKTCPNLESIVRQAV A KKFQQTFVTVPATLRLFFHDCFVQGCDASVLIASTGNNQAEKDHPDNLSLAGDGFDTVIKAKAAVDAIPQCRNKVSCADILALATRDVIALSGGPSYTVELGRFDGLVSRTSDVNGRLPQPTNNLNQLNSLFAANGLTQ TDMIALSGAHTLGFSHCSKFASRIYSTPVDPTLNKQYVAQLQQMCPRNVDPRIAINMDPTTPRKFDNVYYQNLQQGKGLFTSDQILFTDPRSRNTVNSFASSSNVFNSNFVAAMTKLGRVGVKTARNGKIRTDCSVL*.

[0057] Example 4 Soybean GmPOD123 Construction of gene expression vector and stable genetic transformation of soybean

[0058] The pMD18T-GmPOD123 plasmid was used for PCR with homology arm primers, and the pCAMBIA3301-Flag vector plasmid was used Nco Ⅰ and BnE Ⅱ for double restriction digestion, and ligated using ClonExpress II One Step Cloning Kit (C112-01) to obtain the overexpression vector pCAMBIA3301-GmPOD123-Flag (see the map of the overexpression vector for details). Figure 4 );

[0059] The empty vector and the overexpression vector pCAMBIA3301-GmPOD123-Flag were transformed into EHA105 Agrobacterium tumefaciens for stable genetic transformation of soybean to obtain overexpression vector. GmPOD123 Genetically modified soybeans (denoted as OE- GmPOD123 ). Used to detect overexpression GmPOD123 The electrophoresis diagram of genetically modified soybean is shown in Figure 5 (exist Figure 5 In the table, 1-8 correspond to OE- GmPOD123-1 To OE- GmPOD123-8 ); Overexpression GmPOD123 The expression level identification results of transgenic soybeans are shown in Figure 6 .

[0060] according to Figure 6 of GmPOD123 The expression level of the gene was determined by selecting the high expression strain OE- GmPOD123 -4 for subsequent experiments.

[0061] Example 5 Overexpression soybean GmPOD123 Phenotypic identification of genes under drought stress

[0062] The OE- GmPOD123 -4 transgenic lines and wild type WT (Dongnong 50) were subjected to drought stress treatment in the following steps:

[0063] (1) Mix 95 mL of 95% sodium hypochlorite and 5 mL of 36% concentrated hydrochloric acid and apply to soybean seeds (OE- GmPOD123 -4 seeds and WT seeds) were sterilized with chlorine gas overnight;

[0064] (2) Wash the soybean seeds with sterile water and transfer the seeds of each material to a sterile Petri dish (9 cm) containing a double layer of filter paper (serving as a bud bed);

[0065] (3) The experiment was divided into two treatments: drought stress and control.

[0066] 10 mL of sterile deionized water was added to the culture dish of the control treatment to simulate the normal conditions of soybean germination, and three parallel replicates were set up (respectively WT-CK-1, WT-CK-2, WT-CK-3, WT-CK-4; OE-CK-GmPOD123-4-1, OE-CK-GmPOD123-4-2, OE-CK-GmPOD123-4-3, OE-CK-GmPOD123-4-4), with 30 seeds in each replicate;

[0067] 10 mL of a hypertonic solution containing -0.50 MPa PEG-6000 (polyethylene glycol 6000) was added to the culture dish under drought stress to simulate the drought stress condition during soybean germination. Four parallel replicates (respectively denoted as WT-DS-1, WT-DS-2, WT-DS-3, and WT-DS-4; OE-DS- GmPOD123 -4-1、OE-DS- GmPOD123 -4-2, OE-DS- GmPOD123 -4-3, OE-DS- GmPOD123 -4-4), 30 grains per replicate;

[0068] The culture dishes for drought stress and control treatments were placed in the same environment for culture. From the beginning of the germination experiment, new filter paper and culture dishes were replaced in the clean bench every other day. Sterile deionized water was added to the control treatment, and -0.50 MPa PEG-6000 hypertonic solution was added to the drought stress treatment group to prevent bacterial contamination.

[0069] Overexpression was counted starting from the second day of the germination experiment. GmPOD123 The germination number and hypocotyl length of seeds and WT (Dongnong 50) are shown in Figure 7 (exist Figure 7 The length of the ruler is 5 cm) and Figure 8 .

[0070] Combination Figure 7 and Figure 8 It can be seen that overexpression began on the 6th day of the germination experiment GmPOD123 The number of seed germination and hypocotyl length were significantly better than those of WT (Dongnong 50). GmPOD123 Genes play an important role in regulating soybean plants' ability to resist drought stress.

[0071] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Overexpression GmPOD123 The use of a gene in improving drought resistance of a plant during the germination period is characterized in that: Said GmPOD123 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; The plant is soybean; Said GmPOD123 There is an A / G natural variation at position 145 of the gene, and the A / G natural variation is related to drought resistance of soybean during the germination period.

2. The use according to claim 1, characterized in that The drought resistance of soybean during germination period is manifested as follows: under drought stress, the germination rate and hypocotyl length of soybean germplasm containing haplotype I are significantly higher than those of soybean germplasm containing haplotype II; The haplotype I is: soybean germplasm of HapG genotype; The haplotype II is soybean germplasm of HapA genotype.

3. The use according to claim 1, characterized in that Said GmPOD123 The gene cloning method includes the following steps: (1) Extract RNA from soybean plants and obtain cDNA by reverse transcription; (2) using the cDNA as a template and using the primer pair to perform PCR amplification to obtain a gene sequence as shown in SEQ ID NO: 1; The primer pair includes GmPOD123-F and GmPOD123-R; The nucleotide sequence of GmPOD123-F is shown in SEQ ID NO: 2; The nucleotide sequence of GmPOD123-R is shown in SEQ ID NO: 3; The variety of the soybean plant is: Changnong 26.

4. The use according to claim 3, characterized in that The conditions for PCR amplification in step (2) are: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, for a total of 35 cycles, and post-extension at 72°C for 8 min.

5. A method for improving drought resistance of soybeans in the germination period, characterized in that: The steps include: S1. Connect the gene sequence shown in SEQ ID NO: 1 to the starting vector to obtain an overexpression vector; S2, transferring the overexpression vector obtained in step S1 into the starting strain to obtain a recombinant engineered bacterium; S3, infect soybean with the recombinant engineering bacteria obtained in step S2 to obtain overexpression GmPOD123 of genetically modified soybeans.

6. The method according to claim 5, characterized in that The starting vector in step S1 is pCAMBIA3301-Flag vector.

7. The method according to claim 5, characterized in that The starting strain in step S2 is a prokaryotic microorganism or a eukaryotic microorganism.

8. The method according to claim 7, characterized in that The starting strain is EHA105 Agrobacterium.

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