Soybean expansin gene gmexpa10 and application thereof

Abstract

This invention relates to the field of genetic engineering technology, specifically providing a soybean extended protein gene. GmEXPA10 and its applications, the GmEXPA10 The dominant expression in soybean pods is an open reading frame (ORF) of 747 bp, encoding 248 amino acid residues. Further analysis in this invention revealed that... GmEXPA10 Overexpression significantly increased the root length of transgenic Arabidopsis thaliana, with a greater increase under acidic conditions; GmEXPA10 Overexpression significantly increased the length and width of transgenic Arabidopsis thaliana pods; GmEXPA10 Gibberellin expression in pods was induced. Analysis of different soybean varietal resources... GmEXPA10 Allelic variation revealed that a downstream SNP (C / A) can classify soybean resources into two types, with significant differences in 100-seed weight and seed width between the types, indicating that the SNP site is related to soybean seed size and weight.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically providing a soybean extended protein gene. GmEXPA10 And its applications. Background Technology

[0002] Soybeans are an important food and oilseed crop, supplying 69% of the world's plant protein and 27% of its plant oil. They not only provide essential amino acids and unsaturated fatty acids for the human body, but also serve as animal feed for livestock and aquaculture. However, their yield is relatively low compared to other crops, far from meeting demand. Therefore, increasing soybean yield is an important breeding goal in my country at present.

[0003] Expansins are loose proteins located in plant cell walls, playing crucial roles in plant growth and development, yield and quality traits, and resistance to various biotic and abiotic stresses. Currently, expansins are considered the only cell wall relaxants in plants capable of restoring the extensibility of heat-inactivated cell walls, and are key factors regulating acidic growth in plants. Expansins identified in plants are generally classified into four classes: α-expansin (EXPA), β-expansin (EXPB), α-expansin-like proteins (EXLA), and β-expansin-like proteins (EXLB). Experiments have demonstrated that α-expansin and β-expansin play important roles in inducing cell wall relaxation in plants. They have been found to have significant regulatory effects on the normal growth and development of the entire plant, including seed germination, root development, leaf growth, internode elongation, fruit softening, pollen tube elongation, organ abscission, and stomatal opening and closing.

[0004] Currently, there are reports on the functional studies of extended protein genes in soybean root development and nodulation nitrogen fixation. Cho et al. used Arabidopsis mutants to study extended proteins AtEXP7 and AtEXP18, and found that specific expression of these two genes could promote root hair growth in Arabidopsis. Lee et al. found that soybean extended protein GmEXP1 is mainly expressed in the root tip epidermal cells of primary and secondary roots, and overexpression of GmEXP1 in tobacco significantly enhanced root growth. Ren Mingxuan et al. cloned the full-length sequence of the extended protein gene GmEXPA41 from dwarf soybean. Through bioinformatics analysis, they initially believed it might be related to cell elongation. Subsequently, they constructed it into the E. coli expression vector pET32a and transformed it into BL2UDE3. After prokaryotic expression and SDS-PAGE analysis, high-yielding strains were screened, and fermentation culture was carried out in a fermenter, laying the foundation for the subsequent isolation and purification of soybean extended proteins. Wang Duojia et al. cloned the extended protein gene Cs-EXPA2 from cucumber dwarf mutants using RT-PCR. After analyzing the differences in mRNA expression during the 88-hour growth period of the seedlings, they found that Cs-EXPA2 plays an important regulatory role in the phenotype of cucumber dwarf mutants.

[0005] However, there are few reports on the role of soybean extensin genes in plant yield formation. Therefore, studying the function of soybean extensin genes in plant yield formation is of great significance for a comprehensive understanding of their biological functions. Summary of the Invention

[0006] In view of this, the present invention provides an extended protein gene related to soybean yield. GmEXPA10 This provides important functional genes for further improving soybean yield.

[0007] In a first aspect, the present invention provides a soybean extended protein gene. GmEXPA10 The soybean extended protein gene GmEXPA10 The open reading frame sequence is shown in SEQ ID No. 1.

[0008] SEQ ID No.1: ATGGTGCTTTTGGGACTTCTCATGGTGGGATTTCTTTCTCTGGGTTCATTTGTCTCAGCTTCTGGTTATGGTTGGATGGATGCTCATGCAACCTTCTATGGAGGGGGTGATGCCTCAGGCACCATGGGTGGAGCATGTGGATATGGGAACTTGTACAGCCAGGGCTATGGAACAAACACTGCTGCTTTGAGCACAGCTTTGTTCAACAATGGTTTAAGCTGTGGGGCATGTTTTGAGATAAAGTGTGTTAACGACCAAAGGTGGTGCCTTCCACGCTCAGTTATTGTCACTGCAACCAATTTCTGTCCCCCAAACAATGCACTCCCTAATAATGCAGGTGGATGGTGTAACCCTCCCCTTCACCACTTCGACCTTTCCCAACCTATTTTCCAACAAATTGCTCAATACAAAGCTGGAATAGTACCCGTGGCTTACCGAAGGGTCCCTTGCCGAAAGAGAGAGGGCATCAGATTCACCATCAACGGTCACTCATACTTCAACCTAGTCCTAATTTCCAACGTTGGAGGTGCAGGTGATGTTCACGCAGTTTCCATCAAAGGATCAAGAACAAATTGGCAACCAATGACCAGAAACTGGGGTCAGAACTGGCAAAGCAACGCCTATCTCAACGGACAAAGCCTCTCCTTCAAGGTCACCACTAGCGACGGACACACCGTGGTCTCTAATAATGTTGCACCCTCAAGCTGGTCCTTTGGCCAAACCTTCAATGGCCACCAATTCATTTAA。

[0009] The soybean expansin gene GmEXPA10 The encoded amino acid sequence is as shown in SEQ ID No.2.

[0010] SEQ ID No.2: MVLLLGLLMVGFLSLGSFVSASGYGWMDAHATFYGGGDASGTMGGACGYGNLYSQGYGTNTAALSTALFNNGLSCGACFEIKCVNDQRWCLPRSVIVTATNFCPPNNALPNNAGGWCNPPLHH FDLSQPIFQQIAQYKAGIVPVAYRRVPCRKREGIRFTINGHSYFNLVLISNVGGAGDVHAVSIKGSRTNWQPMTRNWGQNWQSNAYLNGQSLSFKVTTSDGHTVVSNNVAPSSWSFGQTFNGHQFI.

[0011] Secondly, the present invention provides the aforementioned soybean extended protein gene. GmEXPA10 Applications in increasing plant root length.

[0012] Thirdly, the present invention provides the aforementioned soybean extended protein gene. GmEXPA10 Applications in increasing the width, diameter, or surface area of ​​plant seeds.

[0013] In the application provided by this invention, the soybean extended protein gene is overexpressed in plant cells. GmEXPA10 Increase soybean extended protein gene GmEXPA10 The expression level of this substance increases the length of plant roots, the width of soybean seeds, the diameter of soybean seeds, or the surface area of ​​soybean seeds.

[0014] Fourthly, the present invention provides a SNP related to soybean seed traits, wherein the SNP is located at position 40942066 on soybean chromosome 4, and the polymorphism of the SNP is C or A.

[0015] Fifthly, the present invention provides the aforementioned soybean extended protein gene. GmEXPA10 Or the application of the aforementioned SNPs in soybean variety identification.

[0016] and the aforementioned soybean extended protein gene GmEXPA10 Or the application of the aforementioned SNPs in soybean variety breeding.

[0017] Sixthly, the present invention provides a method for identifying soybean varieties, obtaining the polymorphism at position 40942066 of chromosome 4 of the soybean to be tested. Compared with the soybean variety with polymorphism AA, the soybean variety with polymorphism CC has a heavier 100-seed weight, larger seed width, seed diameter, and seed surface area.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention provides a novel soybean extended protein gene. GmEXPA10 The GmEXPA10 In soybean pod dominant expression GmEXPA10 Gene overexpression significantly increased the root length of transgenic Arabidopsis thaliana, with a greater increase under acidic conditions; GmEXPA10 Gene overexpression significantly increased the length and width of transgenic Arabidopsis thaliana pods; GmEXPA10 Gene expression in the pods is induced by gibberellin.

[0020] The present invention also provides a location located at GmEXPA10 The downstream SNP (C / A) of the gene is related to soybean seed size and weight; the SNP can be used to classify soybean resources into two types, and there are significant differences between the types in terms of 100-seed weight, seed width, etc. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 This is an analysis of the conserved domains of soybean extended protein GmEXPA10 (red line: signal peptide sequence; black line: catalytic region; green line: binding region).

[0023] Figure 2 It is the subcellular localization of soybean extended protein GmEXPA10.

[0024] Figure 3 It is soybean GmEXPA10 Expression analysis of NDD2 in different tissues and at different stages of pod development; A: Expression analysis of NDD2 in different tissues; B: Expression analysis of ZH13 in different tissues.

[0025] Figure 4 It is a transfer GmEXPA10 Arabidopsis thaliana PCR detection and qRT-PCR analysis; Figure A: PCR detection (M: DNA marker DL2000; 1: wild type; 2-7: overexpression transfection) GmEXPA10 Arabidopsis thaliana); Figure B: qRT-PCR analysis (WT: wild type; OE1~OE3: overexpression transfection) GmEXPA10 Arabidopsis thaliana).

[0026] Figure 5 Is it hyperexpression? GmEXPA10Root length analysis of Arabidopsis thaliana; Figure A: pH 4.5 condition; Figure B: pH 5.8 condition; WT: wild-type control; OE1~OE3: overexpression transgenes GmEXPA10 Homozygous lines of Arabidopsis thaliana.

[0027] Figure 6 Is it hyperexpression? GmEXPA10 Analysis of Arabidopsis thaliana pod length and width; where WT: wild-type control; OE1~OE3: overexpression transgenic GmEXPA10 Homozygous lines of Arabidopsis thaliana.

[0028] Figure 7 It is the soybean extended protein gene. GmEXPA10 Allelic variation analysis, where A: GmEXPA10 Allelic variation analysis (downstream SNPs (C / A) are marked in green and blue respectively; B: Analysis of differences in 100-grain weight and grain area between CC and AA (based on downstream SNP (C / A) typing); C: Analysis of differences in grain width and diameter between CC and AA (based on downstream SNP (C / A) typing).

[0029] Figure 8 It is the soybean extended protein gene. GmEXPA10 Promoter clone electrophoresis diagram, where M: DNA marker DL2000; lane 1: GmEXPA10 Promoter; Lane 2: Blank control.

[0030] Figure 9 It is a transfer GmEXPA10 GUS staining images of different tissues and organs of Arabidopsis thaliana with promoters.

[0031] Figure 10 It is the soybean extended protein gene after GA3 treatment. GmEXPA10 Expression analysis. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0033] The soybeans used in this invention are the superior germplasm C813 (large pods and large grains) and KN7 (small pods and small grains), and the Arabidopsis thaliana used is the Columbia ecotype. Both the soybeans and Arabidopsis thaliana were provided by the Soybean Genetics and Breeding Research Group of Hebei Agricultural University.

[0034] The project on which this invention is based has the following project number: 2023YFD2301500; and its name is: Research and Development and Integrated Demonstration of Wheat-Corn (Soybean) Capacity Improvement Technology in the Huang-Huai-Hai Plain.

[0035] The transfer used in this invention GmEXPA10 The method for analyzing the root length of Arabidopsis thaliana was as follows: seeds from three transgenic Arabidopsis thaliana T3 lines and wild-type plants were placed into 1.5 mL centrifuge tubes, sterilized and washed with ddH2O, disinfected with 75% alcohol for 3-5 min, and sterilized and washed with ddH2O 4-5 times. The Arabidopsis thaliana seeds were planted in 1 / 2 MS medium at pH 5.8 and pH 4.5 respectively using a 1 mL pipette tip and placed in a light-cured culture room. After 9 days, the root length of Arabidopsis thaliana was measured with a ruler.

[0036] The transfer used in this invention GmEXPA10 The method for determining the traits related to Arabidopsis thaliana pods was as follows: Three transgenic Arabidopsis thaliana T3 generation lines and a wild-type control were planted in a light-cultured room, with at least 20 plants retained for each material. After the plants matured, 15 uniformly growing plants were selected from each material, and 30 mature pods from the upper part of each plant were taken and photographed using a high-speed scanner. The pod length, pod width, and other traits were then measured using the Wanshen Automatic Seed Testing and Analysis System.

[0037] The data analysis method used in this invention is as follows: significance testing of the data is performed using Microsoft Excel 2016, and graphs are created using GraphPad Prism8 software.

[0038] The invention used GmEXPA10 The promoter cloning and GUS staining methods were as follows: using the large-pod, large-grain variety C813 as material, its DNA was extracted using the CTAB method and retrieved through the Phytozome website. GmEXPA10 The upstream ~2000 bp sequence was amplified using the designed promoter cloning primers (Table 1) and the above DNA as a template. GmEXPA10 Promoter sequence and construction of extended protein GmEXPA10pro Fusion GUS expression vector pCamG- GmEXPA10pro -GUS was amplified by PCR and DNA sequencing to confirm its sequence correctness; the GUS expression vector was transformed into Arabidopsis Columbia ecotype using the flower-dipping method. After obtaining positive lines through antibiotic screening, GUS staining solution was used to stain at 37℃ for 12 hours, followed by decolorization with anhydrous ethanol for 7-15 days until the anhydrous ethanol became colorless. The lines were then observed and photographed under a stereomicroscope.

[0039] The GA3-induced induction used in this invention GmEXPA10The expression level analysis method was as follows: Ten plants of each of the C813 and KN7 varieties were planted in an artificial climate chamber. After flowering, five plants were grouped together, and their flowers and pods were sprayed with GA3 and H2O (control), respectively, every other day. Subsequently, at 9, 12, and 15 days after flowering, pods were harvested, RNA was extracted, and cDNA was reverse transcribed. qRT-PCR was performed using pre-designed primers (Table 1), and 2... -ΔΔCT The expression level can be calculated using this method.

[0040] Example 1: Extended Protein GmEXPA10 Cloning methods

[0041] Using cDNA from the rapid development stage of C813 pods as a template, open reading frame primers were designed using the online website JustBio (https: / / justbio.com / ) (Table 1) to amplify the DNA. GmEXPA10 The amplified products were detected by agarose gel electrophoresis and DNA sequencing to obtain... GmEXPA10 Open reading frame sequence, as shown in SEQ ID No. 1.

[0042] Table 1 Primers used in this invention

[0043]

[0044] This embodiment uses cDNA from the rapid pod development stage of the superior large-pod, large-seed germplasm C813 as a template to amplify the extended protein gene. GmEXPA10 After recovering the target band and sequencing the DNA, GmEXPA10 The open reading frame is 747 bp. Further findings indicate that... GmEXPA10 Located on chromosome 40 938 439–40 941 582 of the soybean reference genome Williams82 (a4V1), it contains 3 exons and 2 introns, encoding 248 amino acids, as shown in SEQ ID No. 2, with a molecular weight of 26.7 kDa and the molecular formula C. 1190 H 1787 N 335 O 344 S 13 The theoretical isoelectric point is 8.96.

[0045] Example 2 Bioinformatics Analysis of Extended Protein GmEXPA10

[0046] The GmEXPA10 signal peptide sequence was analyzed using SignalIP-5.0 (https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / ), the conserved domains of GmEXPA10 were analyzed using InterPro (https: / / www.ebi.ac.uk / interpro / search / text / ), and homology comparisons of GmEXPA10 and other α-subfamily extended proteins from other species were performed using BioXM 2.7.1 software.

[0047] Bioinformatics analysis revealed that the GmEXPA10 signal peptide sequence is located between amino acids 1 and 20, with the expected cleavage site between amino acids 20 and 21; further analysis of conserved domains revealed ( Figure 1 The protein contains both a catalytic domain and a binding domain, essential for its function. The catalytic domain is located at amino acids 43-155, and the binding domain is located at amino acids 156-244. It was also found to contain two conserved amino acids (cysteine ​​C and tryptophan W), necessary for the function of this type of protein, suggesting that GmEXPA10 binds to the plant cell wall and performs its catalytic function, thereby relaxing the cell wall, releasing intracellular swelling pressure, and causing cell expansion.

[0048] Subcellular localization prediction of GmEXPA10 using online tools revealed that it is located in the plant cell wall; further, its GFP fusion expression vector was constructed, transformed into tobacco leaves, and observed by plasmolysis and laser confocal microscopy, and the results showed that ( Figure 2 ), located in the plant cell wall.

[0049] Using transcriptome data from soybean varieties, we analyzed extended proteins. GmEXPA10 Expression in different tissue sites and at different stages of pod development revealed that ( Figure 3 The expression level of this gene in soybean reproductive organs (such as flowers, pods, and grains) is higher than that in vegetative organs (such as roots, stems, and leaves), indicating that it mainly plays a role in the later stages of growth and development, especially during pod formation and grain filling. Furthermore, the expression level of this gene shows an increasing trend with pod growth and development, indicating that it is closely related to pod growth and development.

[0050] Example 3: Extended Protein GmEXPA10 Transformation of Arabidopsis

[0051] Building extended proteins GmEXPA10The overexpression vector pCHAC-GmEXPA10 was amplified by PCR and its sequence was confirmed to be correct. The overexpression vector was transformed into Arabidopsis thaliana Columbia ecotype using the flower-dipping method. Transgenic Arabidopsis thaliana was identified by antibiotic screening, PCR amplification, DNA sequencing and qRT-PCR until T3 homozygous lines were obtained.

[0052] overexpression GmEXPA10 PCR testing was performed on Arabidopsis thaliana, and the results showed that ( Figure 4 This can amplify the expected target band, allowing for further analysis of transgenic Arabidopsis thaliana. GmEXPA10 Expression levels, the results showed ( Figure 4 ), 3 homozygous transgenic lines GmEXPA10 The expression levels were significantly higher than those of the wild-type control, indicating that... GmEXPA10 It has been integrated into the Arabidopsis genome and can be expressed normally, which can be used for subsequent identification of gene biological functions.

[0053] To understand GmEXPA10 To determine whether it possesses the function of relaxing cell walls and thus causing plant cells to expand, we first analyzed three overexpression transgenes. GmEXPA10 Root-related traits of homozygous Arabidopsis thaliana lines under different pH conditions were studied, and the results showed that ( Figure 5 Under pH 4.5 and pH 5.8 conditions, the root length of all three transgenic Arabidopsis lines was significantly longer than that of the wild-type control, with an increase of 30.5%–35.7% at pH 4.5 and 15.0%–17.9% at pH 5.8. This indicates that the root length of the transgenic Arabidopsis lines was significantly longer than that of the wild-type control. GmEXPA10 It has the function of catalyzing the hydrolysis of non-covalent bonds between cellulose and hemicellulose, thereby relaxing the cell wall, resulting in a significant increase in root length in transgenic Arabidopsis thaliana overexpression; and since the extended protein gene is an acid-condition-inducible gene, the increase is even greater in transgenic Arabidopsis thaliana under slightly acidic conditions (pH 4.5).

[0054] Analysis of overexpression GmEXPA10 The pod length and pod width of homozygous Arabidopsis thaliana lines were observed to be ( Figure 6 The pod length of all three lines was significantly longer than that of the wild-type control, with increases ranging from 8.4% to 19.8%, and the pod width of two lines was also significantly longer than that of the wild-type control, with increases ranging from 7.0% to 19.3%. Therefore, GmEXPA10 It has the function of promoting the growth and development of transgenic Arabidopsis thaliana pods.

[0055] Example 4: Extended Protein GmEXPA10 Allelic variation analysis

[0056] Using the soybean variety resource resequencing data from the research group, we analyzed GmEXPA10 The full-length sequence contained SNP allelic variations, revealing a total of 9 SNPs, including 1 SNP located downstream of the gene and 8 SNPs located within introns. Figure 7 Further analysis of the resequencing varietal resources using the aforementioned nine SNPs revealed that the SNP (C / A) located downstream of this gene (position 40942066 on soybean chromosome 4) can classify the tested varietal resources into two types (CC and AA). Significant or highly significant differences were found between the two types in terms of 100-seed weight, seed width, diameter, and area, suggesting that this SNP is related to soybean seed size.

[0057] Example 5 GmEXPA10 Promoter cloning and expression analysis

[0058] Cloning using superior soybean germplasm C813 GmEXPA10 Upstream promoter sequence ( Figure 8 The transgenic gene was ligated into a GUS-containing expression vector, transformed into Arabidopsis thaliana, and homozygous lines were obtained by molecular biological assays. Different tissues and organs of the transgenic homozygous lines were sampled and stained with GUS. The results showed that... Figure 9 GUS staining was mainly distributed in the reproductive organs of transgenic Arabidopsis, while the staining in vegetative organs was very light, with the deepest GUS staining observed in the pods of transgenic Arabidopsis. GmEXPA10 It mainly plays a role in the reproductive growth stage of plants and is of great significance to the growth and development of plant pods, which is consistent with the results that overexpression of this gene can significantly increase the length and width of transgenic Arabidopsis thaliana pods.

[0059] Meanwhile, through analysis of clones obtained GmEXPA10 Upstream promoter sequences were analyzed, revealing that in addition to common promoter elements (TATA-box, CAAT-box, etc.), they also contained plant hormone response elements such as gibberellin (2 elements), methyl jasmonate (2 elements), and abscisic acid (4 elements), indicating that they may function through these hormone signaling pathways. Further analysis was conducted using gibberellin to treat pods of different soybean varieties at different times after flowering. GmEXPA10 The expression levels were analyzed, and the results showed that the pods of the large-pod, large-seed variety C813 and the small-pod, small-seed variety KN7 were expressed at different times after flowering. GmEXPA10 The expression levels of both increased significantly after gibberellin treatment. Figure 10 The results indicate that the gene is upregulated by gibberellin, and its expression level in the C813 variety is higher than that in the KN7 variety, suggesting that the gene is closely related to the growth and development of soybean pods.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Soy extended protein gene GmEXPA10 Its application in increasing the length and width of plant pods is characterized by, Soy extended protein gene GmEXPA10 The open reading frame sequence is shown in SEQ ID No. 1; or The soybean extended protein gene GmEXPA10 The encoded amino acid sequence is shown in SEQ ID No. 2; The application method involves overexpressing the soybean extended protein gene in plant cells. GmEXPA10 Increase soybean extended protein gene GmEXPA10 The amount of expression; The plant is soybean and / or Arabidopsis thaliana.

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