Peanut ahgapa5 gene and application thereof

Abstract

This invention discloses a peanut AhGAPA5 This invention relates to genes and their applications, belonging to the field of plant gene technology. It yielded 21 varieties of peanuts. GAPDH Genes were identified and experimentally verified. Peanut AhGAPA5 is a GAPDH located in chloroplasts, an isoform of the chloroplast glyceraldehyde-3-phosphate dehydrogenase GAPA, and overexpression of peanut... AhGAPA5 Genes can increase the net photosynthetic rate of plants, thereby improving their drought resistance and yield. This is beneficial for breeding drought-resistant peanut varieties and promoting the industrialization of peanut cultivation. It also provides an important basis for peanut stress-resistant breeding and efficient utilization of calcium fertilizer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genes, and particularly relates to a peanut AhGAPA5 gene and application thereof. BACKGROUND

[0002] Drought stress is a common abiotic challenge, which not only affects the transpiration, photosynthetic efficiency, root stem leaf growth and reproductive development of plants, but also affects some potential physiological processes, including cell division, cell wall mechanics, water transport, primary and secondary metabolism and detoxification of reactive oxygen species. With the intensification of the greenhouse effect, the global environment is deteriorating, the drought area of arable land is increasing, and the effective arable land area is gradually decreasing, which seriously restricts the development of agriculture. Peanut is an important economic crop and oil crop worldwide, which provides rich protein and oil resources for people's daily life. With the increasing demand for peanuts, the supply and demand problem of peanuts is increasingly prominent, and drought is one of the main factors limiting the growth and development of peanuts.

[0003] Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in higher plants is a key enzyme in glycolysis, gluconeogenesis and the Calvin cycle process, and is divided into GAPC, GAPCp and GAPA / B three types of isoenzymes due to different cellular localization. GAPA / B is a GAPDH in chloroplast, which reduces 1,3-diphosphoglycerate to glyceraldehyde-3-phosphate (reverse reaction of glycolysis) with NADPH or NADH as coenzyme, and plays a central role in the Calvin cycle of assimilating CO2. At present, there are few studies on chloroplast GAPDH under abiotic stress. It is of great significance to study how to improve photosynthesis and yield of crops. SUMMARY

[0004] The application aims to provide a peanut AhGAPA5 gene and application thereof. The application obtains 21 peanut GAPDH gene family members through comparison and screening, and explores the chromosomal localization, motif, domain, gene structure and cis-acting elements of the peanut AhGAPDH gene family members, and proves that the peanut AhGAPA5 gene can improve the net photosynthetic rate of plants, which is beneficial to the industrialization development of peanuts.

[0005] To achieve the above application purposes, the application adopts the following technical solutions:

[0006] The application provides a peanut AhGAPA5 gene, wherein the nucleotide sequence of the peanut AhGAPA5 gene is shown as SEQ ID No. 1.

[0007] The application further provides the peanut AhGAPA5The peanut AhGAPA5 protein encoded by the gene, the amino acid sequence of which is shown in SEQ ID No. 2.

[0008] The peanut AhGAPA5 is GAPDH located in chloroplasts and is an isoform of chloroplast glyceraldehyde-3-phosphate dehydrogenase GAPA.

[0009] The present invention also provides a recombinant expression vector containing the aforementioned AhGAPA5 Gene.

[0010] The present invention also provides an engineered bacterium containing the aforementioned... AhGAPA5 Gene.

[0011] The present invention also provides the aforementioned AhGAPA5 The application of genes in improving plant stress resistance and increasing plant yield.

[0012] Furthermore, the method for improving plant stress resistance involves overexpressing the aforementioned [method / ingredient] in plants. AhGAPA5 Genes that promote an increase in the net photosynthetic rate of plants.

[0013] Furthermore, the application of calcium can promote the aforementioned effects under drought stress. AhGAPA5 Gene expression enhances plant stress resistance and yield.

[0014] Furthermore, the plant roots were irrigated with a solution containing 6 mmol / L calcium ions.

[0015] Furthermore, the plant's stress resistance is drought resistance.

[0016] Furthermore, the plants mentioned are Arabidopsis thaliana and peanut.

[0017] The present invention also provides the aforementioned AhGAPA5 Application of genes in breeding drought-resistant peanut varieties.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] This invention obtained 21 varieties of peanuts through screening. AhGAPDH These genes encode proteins, except for AhGAPA4 and AhGAPA9, which are all stable proteins; and except for AhGAPA8, which are all hydrophobic proteins. Experiments have confirmed that 21 genes... GAPDH Genes are located on 12 different chromosomes and have 4 to 10 motifs. AhGAPDH Genes participate in various abiotic stress defense responses and regulate a wide range of life processes.

[0020] This invention has been experimentally verified to show that peanut AhGAPA5 is a GAPDH located in chloroplasts, an isoform of chloroplast glyceraldehyde-3-phosphate dehydrogenase GAPA, and that overexpression of peanut... AhGAPA5 The gene can increase the net photosynthetic rate of Arabidopsis thaliana, thereby improving the plant's stress resistance, which is beneficial to promoting the industrialization of peanuts and has important practical significance. Attached Figure Description

[0021] Figure 1 For peanuts GAPDH The distribution of gene families on chromosomes, with the left-hand scale unit being megabases (Mb).

[0022] Figure 2 For peanuts GAPDH Gene family member analysis, where a represents gene family clustering, b represents MEME software analysis and prediction results, c represents conserved domain prediction results, and d represents gene structure.

[0023] Figure 3 For peanuts GAPDH Predictive analysis of cis-acting elements in gene family promoters.

[0024] Figure 4 For constructing models of peanut, soybean, rapeseed, Arabidopsis, and tobacco based on the ML maximum likelihood method GAPDH Gene family phylogenetic tree.

[0025] Figure 5 This is a sequence analysis diagram of peanut chloroplast GAPDH (GAPA).

[0026] Figure 6 This is a comparative graph showing the phenotypic differences between calcium-deficient and calcium-treated peanut plants after drought treatment.

[0027] Figure 7 For calcium deficiency in peanut leaves and roots after drought treatment and calcium application AhGAPA5 Gene expression diagram, where A represents the relative expression level in leaves; B represents the relative expression level in roots.

[0028] Figure 8 For WT and positive / negative expressions AhGAPA5 Determination of net photosynthetic rate of gene lines. Detailed Implementation

[0029] The technical solution of the present invention will be further described in detail with reference to the following specific examples.

[0030] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0031] Example 1

[0032] The whole genome sequence of peanut (arahy.Tifrunner.gnm2.ann1.4K0L.cds.fna), annotation file (arahy.Tifrunner.gnm2.ann1.4K0L.gene_models_main.gff3) and protein sequence (arahy.Tifrunner.gnm2.ann1.4K0L.protein.faa) were obtained from the Peanut Base (https: / / www.peanutbase.org / ) database. The hidden Markov model files of conserved domains of GADPH gene family were obtained from pfam database (http: / / pfam.xfam.org / ): PF00044 and PF02800. The genome sequence and annotation file of Arabidopsis thaliana ( Arabidopsis thaliana ), Glycine max ( Glycine max ), Brassica napus ( Brassica napus ), Zea mays ( Zea mays ), and Nicotiana attenuata ( Nicotiana benthamiana ) were downloaded from Ensembl plants (http: / / plants.ensembl.org / index.html). The GAPDH protein sequence of Arabidopsis thaliana was downloaded from TAIR (http: / / www.arabidopsis.org / ).

[0033] The prediction information of amino acid number, molecular weight, theoretical pi, instability index, aliphatic amino acid index, and average hydropathy of the protein of AhGAPDH family members was obtained by Protein Paramter Calc tool of TBtools software. The chromosome location map of gene family members was drawn by Gene Location Visualize from GTF / GFF tool of TBtools software to obtain the location information of the family members on the chromosome. AhGAPDH

[0034] ​The motif structure of AhGAPDH was predicted by the MEME (http: / / http: / / meme-suite.org / index.html) website, the number of motifs functional domain was set to 10, and other optional parameters were default values, and the conserved motif of AhGAPDH was detected. The mast.xml result file was downloaded, and the Gen Structure View (Advanced) tool of TBtools software was used to visually analyze the motif, domain and gene structure of AhGAPDH protein from the level of phylogenetic tree. The Plant CARE database (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) was used to predict possible cis-acting elements based on the promoter sequence of gene position information, and TBtools software was used for visualization. AhGAPDH

[0035] The CDS sequences of soybean, rape, Arabidopsis, tobacco were extracted by the Fasta Extract (Recommended) tool of TBtools software, and were translated into proteins using the Batch Translate CDS to Protein module. By using the Arabidopsis GAPDH family protein sequence, homologous alignment was performed by two methods of hmmsearch and BlastP, and the candidate genes of GAPDH family protein were found, and the redundancy was removed to obtain the preliminary GAPDH family members. Multiple sequence alignment was performed by Clustal W in MEGA 7 software, and the GAPDH protein sequences of peanut, soybean, rape, Arabidopsis and tobacco were compared, and according to the alignment results, the maximum likelihood method (Maximum Likelihood Estimation, MLE) was used to construct the phylogenetic tree, and the online tool iTOL (http: / / itol.embl.de) was used to edit the generated developmental phylogenetic tree map.

[0036] 1、Peanut AhGAPDH Identification of gene family and basic physicochemical characteristics

[0037] Taking the peanut GAPDH protein sequence (XM_025750035.3) as reference, homologous alignment was performed by two methods of hmmsearch and BlastP, and the AhGAPDH protein candidate gene was found, and after removing redundancy, 21 GAPDH genes were finally screened in peanut genome, and 21 AhGAPDH ​Genus members. By alignment of NCBI blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and classification of phosphorylated GAPDH proteins in plants: GAPA / B, GAPC, GAPCp, combined with AhGAPDH The genes are named according to the order of their positions on the chromosome (Table 1).

[0038] The amino acid number, molecular weight, isoelectric point pi, instability index, lipid-soluble index, and total average hydrophilic value prediction information of the protein encoded by the genus members were obtained using TBtools, and the results are shown in Table 2: AhGAPDH The amino acid number of the gene family sequence is between 586 (AhGAPA9) and 223 (AhGAPC5); the molecular weight is between 63306.33 kDa (AhGAPA9) and 24220.73 kDa (AhGAPC5); the isoelectric point pi is between 5.32 (AhGAPA8) and 9.4 (AhGAPA4), of which the members with an isoelectric point pi greater than 7 account for two-thirds of the total genus members; the instability index is between 41.61 (AhGAPA4) and 23.08 (AhGAPC7), of which AhGAPA4 (41.61) and AhGAPA9 (41.38) are unstable proteins, and the other genus members are stable proteins; the lipid-soluble index is between 99.1 (AhGAPA8) and 83.5 (AhGAPC5); the total average hydrophilic value is between 0.082 (AhGAPA8) and -0.282 (AhGAPC3), of which the total average hydrophilic value of AhGAPA8 is positive, and the total average hydrophilic value of the other genus members is negative, which are hydrophobic proteins. Therefore, AhGAPA5 are stable and hydrophobic proteins.

[0039] Table 1 Basic information table of peanut AhGAPDH gene family

[0040] ;

[0041] .

[0042] Table 2 Basic information table of peanut AhGAPDH protein

[0043] .

[0044] 2. Chromosomal localization of AhGAPDH family

[0045] In order to study the genomic distribution of AhGAPDH on the chromosome, the GAPDH location distribution of genus members on the chromosome Figure 1). The results showed that 21 GAPDH genes were located on 12 different chromosomes. Among them, chromosome 18 and 20 each contained 3 GAPDH genes, respectively AhGAPCp3 , AhGAPCp4 , AhGAPC7 , AhGAPC8 , AhGAPA8 , AhGAPA9 ; chromosome 6, 7, 10, 16 and 17 each contained 2 GAPDH genes, respectively AhGAPA1 and AhGAPC2 , AhGAPCp1 and AhGAPCp2 , AhGAPC4 and AhGAPC5 , AhGAPA5 and AhGAPC6 , AhGAPA6 and AhGAPA7 ; chromosome 3, 8, 9, 11 and 14 each contained 1 GAPDH gene, respectively AhGAPC1 , AhGAPA2 , AhGAPC3 , AhGAPA3 and AhGAPA4 . It can be seen that AhGAPA5 gene is on chromosome 16.

[0046] 3、 AhGAPDH Gene family motif, domain and gene structure analysis

[0047] According to the results of the phylogenetic tree, the members of the AhGAPDH gene family can be divided into 5 clusters (a in Figure 2 ). The conserved motifs of 21 AhGAPDH proteins were analyzed using MEME software, and 10 motifs were detected, which were designated as 1 to 10, and the prediction results (b in Figure 2 ) are shown as follows: AhGAPDHThe gene family has 4-10 motifs, and all family members have motif 6 and motif 3. The GAPDH family members in the first cluster, the fourth cluster and the fifth cluster are a large type: GAPCp, and the motif structures are highly similar. One family member AhGAPCp2 in the first cluster and two family members AhGAPCp3 and AhGAPCp4 in the fourth cluster contain all the motif types except for the lack of motif 10 at the 3' end. All members of the second cluster (AhGAPC1-AhGAPC8) have the deletion of 5' end motif 8 and 3' end motif 10. Among them, AhGAPC3 has only 4 motifs (motif 6, motif 3, motif 7 and motif 1), AhGAPC4 has only 5 motifs (motif 5, motif 6, motif 3, motif 7 and motif 1), and all the members of the second cluster have the deletion of 5' end motif 8 and 3' end motif 10. AhGAPDH The gene family members contain the least motif types. In the fifth cluster, only one family member AhGAPCp1 is deleted from 5' end motif 8 and 3' end motif 10. In the third cluster, all members have 5' end motif 8. AhGAPA9, AhGAPA8, AhGAPA6 and AhGAPA2 have all 10 motif types. AhGAPA4 lacks motif 6 and motif 4, AhGAPA3 and AhGAPA7 lack motif 7 and motif 1, and AhGAPA5 and AhGAPA1 lack 3' end motif 10.

[0048] The results of the conserved domain prediction show that (as shown in Figure 2 The first cluster and the fourth cluster have high similarity in motif structure, and both have PLN02272 conserved domain, and the fifth cluster has PLN02272 superfamily conserved domain. The second cluster has PLN02272 superfamily, PLN02358 superfamily, NK superfamily and NADPH_Ox superfamily conserved domains. The third cluster has NADPH_Ox superfamily, PLN02237, PLN02237 superfamily and PLN03096 conserved domains.

[0049] The results of the gene structure analysis show that (as shown in Figure 2 d in the description): AhGAPDH The gene family members have structural diversity, most of the members have 1-2 introns, AhGAPC3 、 AhGAPA9, AhGAPA4 and AhGAPA7 no intron,AhGAPCp3 There are 3 introns, and the number of introns is the most AhGAPC6 , there are 4 introns. In summary, although there are some differences in the structure of AhGAPDH members, except for AhGAPC3 and AhGAPC6 in cluster II, the structural differences between most AhGAPDH family members in the same branch and cluster are small.

[0050] From the above, the structural differences between cluster II and cluster III are more obvious, but the similarity between cluster I and cluster IV in motif, domain and structure is very high. It can be seen AhGAPA5 Through the relatively conserved functional domain, the function of the other AhGAPDH family members are connected to each other, and the structural and sequence changes of the different branch categories of the differentially expressed genes will lead to the differentiation of the function.

[0051] 4、 AhGAPDH Analysis of cis-acting elements of gene promoters

[0052] In order to further study the possible transcriptional regulation mechanism of peanut GAPDH, the upstream 2000bp promoter region of 21 AhGAPDH genes was analyzed.

[0053] The results show that Figure 3 , a total of 29 kinds of cis-acting elements were detected, and these cis-acting elements are mainly related to light response, abscisic acid response, auxin response, gibberellin response, low temperature response, MeJA response, drought induction, anaerobic induction, defense and stress response, etc. Among them, the light response element is the most abundant, followed by the plant hormone response and the non-biological stress response related cis-acting elements. These elements play an important role in the response of plant non-biological stress, indicating that peanut GAPDH is involved in different non-biological stress defense response and regulation of various life processes, and the transcription factor controls transcription by recognizing specific DNA sequences in the promoter region of the gene, guiding genome expression, thereby regulating plant stress resistance, AhGAPA5 The promoter region of AhGAPA5 contains a variety of functional cis-acting elements such as light response, drought response and ABA response, indicating that increasing the activity of AhGAPA5 is of great significance for improving photosynthesis and resisting non-biological stress in crops.

[0054] 5, AhGAPDH family phylogenetic tree analysis

[0055] In order to analyze the evolutionary relationship of GAPDH protein in different species, GAPDH proteins in peanut, soybean, rape, Arabidopsis and tobacco were selected, and protein alignment and evolutionary analysis of the development tree were carried out by MEGA software.

[0056] According to the branching and referring to the evolutionary relationship of GAPDH family members in Arabidopsis and soybean, the results show that the GAPDH proteins are divided into four different branches. AhGAPC3 is alone in the first branch, and the other GAPDH protein family is divided into three branches of GAPA / B, GAPC and GAPCp. Among them, AhGAPA1, AhGAPA2, AhGAPA3, AhGAPA4, AhGAPA5, AhGAPA6, AhGAPA7, AhGAPA8, AhGAPA9 and GAPA / B are classified into one class, which is the second branch; AhGAPC1, AhGAPC2, AhGAPC4, AhGAPC5, AhGAPC6, AhGAPC7, AhGAPC8 and GAPC are classified into one class, which is the third branch; AhGAPCp1, AhGAPCp2, AhGAPCp3, AhGAPCp4 and GAPCp are classified into one class, which is the fourth branch. Figure 4 According to the above branching results, AhGAPA5 protein is in the second branch, which provides an important basis for the function prediction of peanut AhGAPA5 protein.

[0057] The application further proves that the peanut chloroplast GAPDH (GAPA) can interact with calcium-dependent protein kinase, and the structure analysis of the peanut GAPA protein shows that the N-terminal exists chloroplast transduction peptide ( Figure 5 ), and the results of the phosphorylation differential expression screening also show that the peanut AhGAPA5 is a GAPDH located in the chloroplast, which is a chloroplast glycerol-3-phosphate dehydrogenase GAPA isoform.

[0058] Example 2, exploring the response of AhGAPA5 gene to drought stress and calcium in peanut

[0059] The peanut variety Huayu 22 is selected, the seeds are sterilized with 75% alcohol for 30s, repeatedly washed with deionized water for 3-5 times, soaked in sterile water for 3 hours, then planted in the mixed soil of nutrient soil and vermiculite (2:1) in a culture dish under dark conditions for 2 days of germination, then selected the seedlings with consistent growth and took out the roots, washed, and used the Hogland culture solution containing calcium (SC, calcium ion concentration is 6mmol / L) and not containing calcium (NC) for water culture of peanut seedlings, the temperature is 25℃, the light / dark cycle is 16h / 8h. After 10 days, the wild type plants and the overexpression AhGAPA5 gene plants are treated with 20% PEG-6000 solution to simulate drought stress for 2 days, combined with the results shown in Figure 6 , the results show that the wild type plants WT are more sensitive to drought stress than the overexpression AhGAPA5 gene plants, with serious leaf curling and wilting symptoms, while the overexpression AhGAPA5 gene plants have obvious drought resistance.

[0060] To investigate AhGAPA5 whether the response to stress, this embodiment on foot calcium (SC) and calcium deficiency (NC) hydroponic culture of peanut flower 22 flower seedlings were drought treatment (DSC and DNC) for different time, by real-time quantitative PCR experiment, analysis AhGAPA5 gene expression in peanut leaves and roots under drought stress.

[0061] As Figure 7 shown, the results show AhGAPA5 induced expression under drought stress. In leaves, AhGAPA5 the transcript accumulates most after 1 hour of DSC and DNC treatment, and at the peak, the expression of the gene induced by drought stress in DSC leaves is higher than that in DNC. In roots, AhGAPA5 the transcript accumulates most after 2 days of DSC and DNC treatment, and at the peak, the expression of the gene induced by drought stress in DSC roots is still higher than that in DNC. These results show AhGAPA5 participate in the response of peanut to drought stress, and calcium signaling plays an important role in the regulation of drought tolerance in peanut.

[0062] Example 3: Preliminary analysis of Arabidopsis thaliana AhGAPA5 isoforms and functions

[0063] Arabidopsis thaliana seeds of wild type and overexpression AhGAPA5 genes were sterilized with 70% alcohol for 30s, washed repeatedly with deionized water for 3-5 times, soaked in sterile water for 3 hours, then placed in a culture dish for half immersion, planted on MS medium, and after germination, the medium on the roots was washed away, and the seedlings with consistent growth were planted in mixed soil of nutrient soil and vermiculite (2:1), and the seedlings were subjected to drought stress simulated by PEG-6000 solution with a final concentration of 20%.

[0064] The net photosynthetic rate (Pn) of the 3rd fully expanded functional leaf under the growing point was determined by a portable photosynthesis rate meter (Li2Cor6400, USA Li2Cor company), and 10 leaves were selected as replicates for each treatment. P

[0065] After drought stress treatment, the net photosynthetic rate of wild type (WT) and overexpression AhGAPA5 Arabidopsis thaliana lines decreased, compared with antisense expression lines (R5, R8) and wild type (WT), the positive expression AhGAPA5 lines (OE6, OE10) decreased to a lesser extent, which could improve the net photosynthetic rate (Pn). Figure 8

[0066] The above experimental results prove that overexpression of the AhGAPA5 ​​The gene can improve the net photosynthetic rate of the plant, and the application of calcium solution can promote the plant to improve the gene expression under drought stress AhGAPA5 The gene expression, in turn, improves the drought resistance of the plant, improves the yield of the plant, is beneficial to the cultivation of drought-resistant peanut varieties, and is beneficial to the industrial cultivation of peanuts, and provides an important basis for peanut stress breeding and efficient utilization of calcium fertilizer.

[0067] The above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, the technical solutions recorded in the foregoing examples can still be modified or some technical features can be replaced by equivalents for ordinary skilled persons in the art; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. Application of peanut AhGAPA5 gene in improving plant stress resistance and increasing plant yield, characterized in that, The nucleotide sequence of the AhGAPA5 gene is shown as SEQ ID No. 1, the method for improving the stress resistance of plants is overexpressing the AhGAPA5 gene in plants, promoting the net photosynthetic rate and drought resistance of plants; applying calcium can promote the expression of the AhGAPA5 gene under drought stress, improve the stress resistance and yield of plants, and the plants are Arabidopsis thaliana and peanuts.

Images