大豆GmPDAT1参与油脂合成和非生物胁迫应答的功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-1171

摘要/Abstract

摘要：

解析大豆GmPDAT1在油脂合成和非生物胁迫应答中的功能，为大豆油脂改良和抗逆性分子育种提供新的科学参考。应用组学工具鉴定GmPDAT1，运用实时荧光定量PCR分析GmPDAT1在大豆不同组织和3种非生物胁迫的表达模式。使用酵母（Saccharomyces cerevisiae）TAG缺陷型突变体H1246检测GmPDAT1酶活性。结果表明，全基因组鉴定获得6个大豆GmPDAT1基因家族成员（GmPDAT1-A-GmPDAT1-F），除GmPDAT1-F具有8个外显子，其余5个GmPDAT1基因含6个外显子。GmPDAT1启动子区存在多个逆境胁迫响应顺式元件。GmPDAT1编码的蛋白序列长度介于582-668 aa，等电点（pI）为5.91-8.59。GmPDAT1蛋白均具有PLN02517超家族蛋白结构域，为膜结合蛋白，二级结构主要元件为α-螺旋和无规则卷曲。6个GmPDAT1蛋白聚类分为3个亚组，分别与花生AhPDAT1、小桐子JcPDAT1和蓖麻RcPDAT1-2亲缘关系较近。GmPDAT1基因家族成员具有组织特异性表达特性，其中GmPDAT1-B在不同组织均表达，且在发育种子表达量最高。推测GmPDAT1-B可能参与大豆种子TAG合成。酵母（Saccharomyces cerevisiae）TAG缺陷型突变体H1246进行功能互补测试证明，GmPDAT1-B具有催化TAG合成的酶活性。在低温、干旱和盐胁迫处理下，GmPDAT1基因家族成员呈现了不同的表达模式，预示它们可差异化参与大豆不同胁迫应答。尤其是GmPDAT1-B可介导3种不同逆境胁迫的响应。GmPDAT1-B可能具有促进大豆油脂合成和胁迫抗性的双重功能。

关键词: 大豆, 磷脂：二酰甘油酰基转移酶1（PDAT1）, 表达分析, TAG合成, 非生物胁迫

Abstract:

The present study was conducted to investigate functions of GmPDAT1（phospholipid: diacylglycerol acyltransferase）genes in soybean oil biosynthesis and abiotic stress response, providing a new scientific reference for soybean oil improvement and stress resistance molecular breeding. The omics tools were employed to GmPDAT1 genes in soybean. Quantitative real-time RT-PCR was used to examine expression patterns of GmPDAT1 genes in various tissues and under three abiotic stresses. Functional complementarity assay was performed with TAG-deficient yeast（Saccharomyces cerevisiae）mutant H1246. Six soybean GmPDAT1 gene family members（GmPDAT1-A-GmPDAT1-F）were identified. GmPDAT1-F had eight exons while the remaining five GmPDAT1 genes contained six exons. Multiple stress-responsive cis- elements were detected in GmPDAT1 promoter regions. The sequence length of GmPDAT1-encoded proteins was between 582-668 aa and their isoelectric point（pI）proteins ranged from 5.91 to 8.59. All GmPDAT1 proteins had PLN02517 superfamily protein domains, classifying as the membrane-binding proteins. The secondary structure of GmPDAT1 proteins mainly consisted of α-helix and ranclom coiled coil. The six GmPDAT1 proteins were phylogenetically clustered into three subgroups, which were closely related to Arachis hypogaea AhPDAT1, Jatropha curcas JcPDAT1 and Ricinus communis RcPDAT1-2, respectively. The members of GmPDAT1 gene family had tissue-specific expression patterns. Of them, GmPDAT1-B was expressed in various tissues with the highest expression in developmental seeds, suggesting that GmPDAT1-B may function crucially for TAG biosynthesis in soybean seeds. Functional complementarity assay using TAG-deficient yeast（Saccharomyces cerevisiae）mutant H1246 evidenced that GmPDAT1-B had the high enzyme activity to catalyze TAG synthesis. Under the treatment of low temperature, drought and salt stress, GmPDAT1 gene members demonstrated different expression patterns, indicating that they differentially participated in different stress responses in soybean. In particular, GmPDAT1-B possibly mediates soybean responses to the three different stresses. In conclusion, GmPDAT1-B may have dual functions in promoting soybean oil synthesis and stress resistance.

Key words: soybean（Glycine max）, phospholipid: diacylglycerol acyltransferase1（PDAT1）, expression analysis, TAG biosynthesis, abiotic stress

苗淑楠, 高宇, 李昕儒, 蔡桂萍, 张飞, 薛金爱, 季春丽, 李润植. 大豆GmPDAT1参与油脂合成和非生物胁迫应答的功能分析[J]. 生物技术通报, 2023, 39(2): 96-106.

MIAO Shu-nan, GAO Yu, LI Xin-ru, CAI Gui-ping, ZHANG Fei, XUE Jin-ai, JI Chun-li, LI Run-zhi. Functional Analysis of Soybean GmPDAT1 Genes in the Oil Biosynthesis and Response to Abiotic Stresses[J]. Biotechnology Bulletin, 2023, 39(2): 96-106.

图/表 10

表1 引物信息

Table 1 Primer information

引物名称Primer name	引物序列 Sequence（5'-3'）
GmActin-F	AGACCTTCAATGTGCCAGCCA
GmActin-R	CACGACCAGCAAGATCCAACC
qGmPDAT1-A-F	TGGCACCTTTGGGGAATTGT
qGmPDAT1-A-R	TCCTCATACCCAATGCGAGC
qGmPDAT1-B-F	CCCCCAAGATGATGAAGCGT
qGmPDAT1-B-R	AGGGATCCCAACCCCATACA
qGmPDAT1-C-F	GCCGGTTCTAAGTTCAGGCT
qGmPDAT1-C-R	CGTGAGCACCACTTTGTGTG
qGmPDAT1-D-F	ACGGAGAAAAGGGTCGGAAC
qGmPDAT1-D-R	CAAAGTGCAAATGCACCCCA
qGmPDAT1-E-F	CTGCCATGTTGCTTGGCTTT
qGmPDAT1-E-R	TGAAAAGGCCCTCTGCACAA
qGmPDAT1-F-F	TCTTGAGCGAGTTATGCGGG
qGmPDAT1-F-R	ATCAGTCCACACAGCCTCAC
GmPDAT1-B-CDs-F	ATGTCGTTTATACGACGCAGAA
GmPDAT1-B-CDs-R	CTAGAGCTTCAAATTGATCTTTTCA
GmPDAT1-B-pYES2-F	GGGGTACCATGTCGTTTATACGACGCAGAA
GmPDAT1-B-pYES2-R	CGGAATTCCTAGAGCTTCAAATTGATCTTTTCA

图1 GmPDAT1和AtPDAT1蛋白Motif预测（A）和基因结构（B）分析

Fig. 1 Motif prediction（A）and gene structure（B）analysis of GmPDAT1 and AtPDAT1 proteins

表2 大豆 Gm PDAT1蛋白质理化性质

Table 2 Physicochemical properties of GmPDAT1 proteins in G. max

蛋白 Protein	氨基酸数目 Number of amino acids	分子质量 Molecular weight/kD	理论等电点 Theoretical pI	不稳定系数 Instability index	亲水系数 GRAVY	跨膜结构 Transmembrane structure
GmPDAT1-A	668	74.833	8.59	36.12	-0.270	1
GmPDAT1-B	668	74.776	8.59	36.87	-0.256	1
GmPDAT1-C	676	75.571	6.28	41.96	-0.360	1
GmPDAT1-D	668	74.557	6.28	42.50	-0.335	1
GmPDAT1-E	625	70.376	6.45	42.51	-0.173	1
GmPDAT1-F	582	65.783	5.91	44.95	-0.112	1
AtPDAT1	671	74.156	6.50	35.95	-0.301	1

图2 大豆GmPDAT1蛋白的二级结构（A）和三级结构（B）

Fig. 2 Secondary structures（A）and tertiary structures（B）of soybean GmPDAT1 proteins

图3 大豆GmPDAT1蛋白氨基酸序列比对 Ⅰ：盖子区域；Ⅱ：盐桥区域；Ⅲ，Ⅳ和Ⅴ：催化三联体结构。图中保守区域用红框标出

Fig. 3 Amino acid sequence alignment of GmPDAT1 proteins in G. max Ⅰ: Lid domain. Ⅱ: Salt bridge. Ⅲ, Ⅳ and Ⅴ: Catalytic triad. Conservative regions in the figure are marked with red boxes

图4 大豆GmPDAT1与其他物种PDAT1的系统进化树 Gm：大豆；At：拟南芥；Rc：蓖麻；Ah：花生；Si：芝麻；Bn：甘蓝型油菜；Cs：亚麻荠；Oep：油橄榄；Jc：小桐子。颜色由红向绿代表亲缘关系由近到远

Fig. 4 Phylogenetic tree of soybean GmPDAT1s and PDA-T1s from other plant species Gm: Glycine max. At: Arabidopsis thaliana. Rc: Ricinus communis. Ah: Arachis hypogaea. Si: Sesamum indicum. Bn: Brassica napus. Cs: Camelina sativa. Oep: Olea europaea. Jc: Jatropha curcas. The color from red to green indicates the kinship from near to far

图5 大豆GmPDAT1启动子的顺式作用元件 A:顺式作用元件的数量；B：顺式作用元件的分布

Fig. 5 Cis-acting element identified in the promoters of soybean GmPDAT1 genes A: The number of cis-acting elements. B: Distribution of cis-acting elements

图6 GmPDAT1在大豆不同组织中的特异性表达

Fig. 6 Specific expressions of GmPDAT1 in different tissues of soybean

图7 GmPDAT1-B的酵母互补功能验证 A：转基因酵母总油脂含量（INVSc1：野生型酵母；EV：H1246转pYES2空载体；GmPDAT1-B：H1246 转pYES2-GmPDAT1-B）；B：转基因酵母油脂的薄层层析（1：TLC标样；2：H1246；3：转 pYES2空载的H1246；4：野生型酵母NVSc1；5：pYES2-GmPDAT1-B转化的H1246）

Fig. 7 Complementary function assay of GmPDAT1-B gene using yeast mutant H1246 A: Total oil content of transgenic yeast（INVSc1: wild-type yeast. EV: H1246 transformed with pYES2 empty vector. GmPDAT1-B: H1246 transformed with pYES2-GmPDAT1-B）. B: Thin layer chromatography of transgenic yeast oil（1: TLC standard. 2: H1246. 3: H1246 transformed with pYES2 empty vector. 4: Wild-type yeast NVSc1. 5: H1246 transformed with pYES2-GmPDAT1-B）

图8 大豆GmPDAT1在不同胁迫处理下的表达谱

Fig. 8 Relative expressions of soybean GmPDAT1 under different stress treatments

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