大豆扩展蛋白基因GmEXLA1在荚果发育中的功能鉴定

doi:10.13304/j.nykjdb.2024.0111

摘要/Abstract

摘要：

扩展蛋白是一类通过松弛细胞壁释放胞内膨胀压力使植物细胞得以扩张的蛋白，在植物生长发育以及抵御各种逆境胁迫中发挥重要功能。然而，有关扩展蛋白在荚果发育中的作用尚不明确，鉴于此，克隆大豆扩展蛋白基因GmEXLA1，并研究其在荚果发育中的功能。结果发现，GmEXLA1在大豆荚粒中优势表达，开放阅读框长度780 bp，编码259个氨基酸，其中第1~19位氨基酸属于信号肽序列，具有扩展蛋白发挥其生物学功能所必需的2个保守结构域；烟草叶片亚细胞定位发现，编码蛋白GmEXLA1位于植物细胞壁；进一步分析发现，GmEXLA1超表达极显著增加转基因拟南芥的果荚长度、宽度和种子重量，其中种子重量的增加幅度为61.2%~80.6%；而GmEXLA1突变则显著降低大豆的荚长、粒长、粒宽和粒重等性状，其中粒长、粒宽和粒重的降低幅度分别为13.5%、7.0%和25.6%，说明GmEXLA1在植物荚果发育过程中发挥重要作用。另外，分析不同大豆品种资源GmEXLA1等位变异发现15个SNPs，其中位于上游的SNP（A/G）在野生大豆与栽培大豆中的分布频率（A85% vs A38%；G15% vs G62%）存在极显著差异。研究结果为通过生物育种手段改良大豆产量性状提供了重要的扩展蛋白基因。

关键词: 大豆, 扩展蛋白, 细胞壁, 荚果发育, 等位变异

Abstract:

Expansin is a kind of protein that can expand the plant cells by loosening the cell wall and releasing the intracellular expansion pressure. Expansin plays an important role in plant growth， development and resistances to diverse stresses. However， the role of expansin in pod and seed development remains unclear. In view of this， an expansin gene GmEXLA1 was cloned and analyzed in this study， and the gene function in pod and seed development was studied. The results showed that GmEXLA1 was predominantly expressed in soybean pod and seed， with an open reading frame length of 780 bp which encoding 259 amino acids. The amino acids from 1 to 19 were the signal peptide of the expansin GmEXLA1， and GmEXLA1 possessed 2 conserved domains of expansins which were necessary for their expanding functions. The subcellular localization analysis showed that the expansin GmEXLA1 was localized in plant cell wall. Further analysis showed that the overexpression of GmEXLA1 significantly increased the pod length， width and seed weight of transgenic Arabidopsis， with the seed weight increased by 61.2%~80.6%. On the contrary， the nonsense mutation of GmEXLA1 significantly reduced soybean pod length， seed length， width and weight， with the decreases of seed-length， -width and -weight 13.5%， 7.0% and 25.6%， respectively. These results indicated that the expansin gene GmEXLA1 played an important role in plant pod and seed developmental processes. In addition， the allelic variation analysis of GmEXLA1 showed 15 SNPs in different soybean germplasms， with an upstream SNP （A/G） presenting significantly different distribution frequencies between the wild and cultivated soybeans （A85% vs A38%； G15% vs G62%）. Above results provided important functional expansin gene for the improvement of soybean yield related traits via the biological breeding technology.

Key words: soybean, expansin, cell wall, pod and seed development, allelic variation

中图分类号:

S565.1

李娜, 张华, 邢馨竹, 邵振启, 杨占武, 李喜焕, 张彩英. 大豆扩展蛋白基因GmEXLA1在荚果发育中的功能鉴定[J]. 中国农业科技导报, 2025, 27(3): 49-59.

Na LI, Hua ZHANG, Xinzhu XING, Zhenqi SHAO, Zhanwu YANG, Xihuan LI, Caiying ZHANG. Function Analysis of Soybean Expansin Gene GmEXLA1 inPlantPod and Seed Development[J]. Journal of Agricultural Science and Technology, 2025, 27(3): 49-59.

图/表 9

表1 本研究所用引物序列

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence （5’-3’）	引物用途 Primer usage
GmEXLA1-F	GCAGTCGACATGGCTTTCTTTATTCTCTCC	扩增开放阅读框；检测转基因拟南芥；检测大豆突变体 Open reading frame amplification； detection of transgenic Arabidopsis and soybean mutant
GmEXLA1-R	TATGGATCCTGTCCCATCATCGCAGGG
GmEXLA1-RT-F1	TGGCCGTTCGAGTTGAAGAA	转基因拟南芥实时定量分析 Real-time quantitative analysis of transgenic Arabidopsis
GmEXLA1-RT-R1	ATACTGCCCCGTGGTTTCTG
GmEXLA1-Mu-F	GCTGGATCCATGTGTGATCGCTGCT	gmexla1突变体目的基因原核表达 Prokaryotic expression of target gene in gmexla1 mutant
GmEXLA1-Mu-R	TTAGAGCTCTCATGTCCCATCATCGCAG
GmEXLA1-RT-F2	ACTGAAGCTTGGCATTGCTG	gmexla1突变体实时定量分析 Real-time quantitative analysis of gmexla1 mutant
GmEXLA1-RT-R2	CATACTGCCCCGTGGTTTCT
GmActin11-F	ATCTTGACTGAGCGTGGTTATTCC
GmActin11-R	GCTGGTCCTGGCTGTCTCC

图1 大豆扩展蛋白GmEXLA1保守结构域分析注：SP—信号肽；Expansin-like EG45—催化区；Expansin-like CBD—结合区；*为保守半胱氨酸C和色氨酸W。

Fig. 1 Conserved domain analysis of GmEXLA1Note： SP—Signal peptide； Expansin-like EG45—Catalytic domain； Expansin-like CBD—Binding domain；* indicates conserved cysteine and tryptophan.

图2 大豆扩展蛋白GmEXLA1亚细胞定位

Fig. 2 Subcellular localization of GmEXLA1

图3 超表达转GmEXLA1拟南芥种子萌发情况

Fig. 3 Germination of transgenic Arabidopsis with GmEXLA1 over-expression

图4 超表达GmEXLA1拟南芥荚长、荚宽与种子重量分析注：WT—野生型；OE1~OE2—超表达转GmEXLA1拟南芥纯合株系；**表示P<0.01水平差异显著。

Fig. 4 Pod length， pod width and seed weight per plant of Arabidopsis over-expressed GmEXLA1Note： WT—Wild-type； OE1~OE2—Over-expression lines with GmEXLA1； ** indicates significant difference at P<0.01 level.

图5 大豆gmexla1突变体分子检测及其突变位置A：PCR检测，M—DNA marker DL2000，1—Williams 82，2—突变体，3—空白对照；B：PCR产物测序结果，黄色背景标出突变碱基C/T；C：突变体目的基因qRT-PCR检测；D：gmexla1及其突变基因的原核诱导表达蛋白检测，M—蛋白分子量标准，1—诱导前的Williams82的GmEXLA1，2—诱导后的Williams82的GmEXLA1，3—诱导前的突变体gmexla1，4—诱导后的突变体gmexla1；E：gmexla1突变位置

Fig. 5 Molecular identification of soybean gmexla1 mutant and its mutation positionA: PCR identification of soybean gmexla1 mutant， M—DNA marker DL2000, 1—Williams 82， 2—gmexla1 mutant, 3—Blank control; B: DNA sequencing result of PCR amplification， the mutant C/T was marked in yellow background; C: qRT-PCR identification of soybean gmexla1 mutant; D: Detection of induced protein in soybean gmexla1 mutant， M—Protein marker， 1—GmEXLA1 in Williams 82 before induced， 2—GmEXLA1 in Williams 82 after induced， 3—gmexla1 in mutant before induced， 4—gmexla1 in mutant after induced; E: gmexla1 mutation position

图6 大豆gmexla1突变体豆荚长度分析注：WT—Williams 82野生型；Mutant—gmexla1突变体；**表示P<0.01水平差异显著。

Fig. 6 Comparison of pod length between Williams82 and its gmexla1 mutantNote： WT—Williams 82 wild-type control； Mutant— gmexla1 mutant； ** indicates significant difference at P<0.01 level.

图7 大豆gmexla1突变体籽粒长度、宽度和百粒重分析注：**表示P<0.01水平差异显著。

Fig. 7 Comparison of seed-length， -width and hundred-seed weight between Williams82 and its gmexla1 mutantNote： ** indicates significant difference at P<0.01 level.

图8 GmEXLA1等位变异及其在野生和栽培大豆中的分布频率分析A：GmEXLA1等位变异分析，上游启动子SNP为A/G变异；B~E：AA型与GG型大豆相关性状差异分析，依据上游SNP（A/G）分型，*和**分别表示P<0.05和P<0.01水平差异显著；F：上游启动子SNP（A/G）在野生大豆与栽培大豆中的分布频率

Fig. 8 SNPs and distribution frequency of GmEXLA1 in wild and cultivated soybeansA: SNPs in GmEXLA1， the SNP in promoter region is A/G variation; B~E: AA- and GG-type related traits， based of on (upstream SNP (A/G)), * and ** indicate significant difference at P<0.05 and P<0.01 levels, respectively; F: Distribution frequency of upstream SNP (A/G) in wild and cultivated soybeans;

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