大豆VAS1基因家族的鉴定及参与侧根发育的研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-0690

摘要/Abstract

摘要：

拟南芥VAS1基因编码一个磷酸吡哆醛依赖性氨基转移酶，可将吲哚丙酮酸转化为吲哚乙酸的生物合成前体色氨酸，是生长素代谢调控中的一个关键酶。对大豆VAS1基因家族进行全基因组鉴定与表达模式分析，并探究其在根系发育中的作用，为深入挖掘大豆VAS1基因的功能奠定基础。运用生物信息学方法对该基因家族成员进行鉴定和分析，采用实时荧光定量分析该家族在不同逆境处理下的表达模式，克隆GmVAS1-1，进一步研究其功能，并通过酵母双杂交试验筛选大豆GmVAS1-1蛋白的互作因子。结果表明，大豆VAS1基因家族共有2个成员，命名为GmVAS1-1和GmVAS1-2。系统进化树、基因结构和保守基序分析表明，大豆VAS1与豆科植物亲缘关系更近。启动子序列分析发现多个逆境和光响应元件。表达模式分析表明，大豆VAS1家族基因在不同种子发育时期的胚乳中表达量较高。荧光定量PCR分析表明，在干旱和盐胁迫下，大豆根系VAS1基因表达量显著上调。拟南芥过量表达GmVAS1-1植株侧根数较野生型显著减少。酵母互作试验及预测共筛选到17个与GmVAS1-1互作的蛋白。大豆VAS1基因家族成员在胚乳中表达量较高，且响应多种逆境及营养胁迫，其中，GmVAS1-1参与侧根发育。

关键词: 大豆, 生长素, 氨基转移酶VAS1, 非生物胁迫, 根发育

Abstract:

Arabidopsis VAS1 gene encodes a pyridoxal-phosphate-dependent aminotransferase, which regulates the auxin amount through converting the indole pyruvic acid to tryptophan. Genome-wide characterization and expression pattern analysis of soybean aminotransferase VAS1 was conducted to explore their roles in root development, for laying foundation in further mining biological functions of VAS1 in soybean plant（Glycine max）. Bioinformatics approaches were used to identify and analyze the gene members of the soybean VAS1 family, and then fluorescence quantitative analysis was used to analyze the expression pattern of this gene family under different stress treatments. In addition, GmVAS1-1 gene was cloned for further functional analysis. The interaction factors of GmVAS1-1 were screened through yeast two-hybrid method. The results showed that there were two members in the soybean VAS1 gene family, and were named as GmVAS1-1 and GmVAS1-2. Phylogenetic tree，gene structure and conserved motifs analysis indicated that soybean VAS1 was more closely related to legumes（Phaseolus vulgaris，Medicago truncatula，Vigna unguiculata，Lotus corniculatus and Cicer arietinum）. There were multiple stress or light response elements in the promoter region. Expression pattern analysis showed that soybean VAS1 genes were highly expressed in the endosperm at the different seed development stages. Fluorescent quantitative PCR analysis revealed that the expression of GmVAS1 was significantly up-regulated in soybean roots under drought or salt stress condition. The number of lateral roots in overexpressed GmVAS1-1 Arabidopsis was significantly less than that of wild type, suggesting that GmVAS1-1 was involved in regulating lateral root development in plants. A total of 17 proteins interacting with GmVAS1-1 were screened by yeast two-hybrid assays and network prediction, which laid a molecular theoretical basis for further exploring the function and mechanism of GmVAS1-1 gene. In summary, soybean VAS1 gene family are highly expressed in the endosperm and respond to various abiotic and nutritional stresses, among which GmVAS1-1 is involved in the development of lateral root.

Key words: soybean, auxin, aminotransferase VAS1, abiotic stress, root development

杨春洪, 董璐, 陈林, 宋丽. 大豆VAS1基因家族的鉴定及参与侧根发育的研究[J]. 生物技术通报, 2023, 39(3): 133-142.

YANG Chun-hong, DONG Lu, CHEN Lin, SONG Li. Characterization of Soybean VAS1 Gene Family and Its Involvement in Lateral Root Development[J]. Biotechnology Bulletin, 2023, 39(3): 133-142.

图/表 12

表1 引物序列

Table 1 Primer sequence

引物名称Primer name	引物序列Primer sequence（5'-3'）
GmVAS1-AscI-F	GGCGCGCCCTCCCGCAGTCCGTCTCT
GmVAS1-BamHI-R	GGATCCATGGGTTCGTTCGTGAAG
AT1G80360-LP	TCTTGAATCCAATTCCGTGAC
AT1G80360-RP	CTTAACGTTCTCCTTCCCCAG
LB2	GCTTCCTATTATATCTTCCCAAATTACCAATACA
AtActin-qPCR-F	TACCGAGGCTCCTCTTAACC
AtActin-qPCR-R	AGCTTGGATGGCGACATACA
AT1G80360-qPCR-F	CCAAGTGGCACCTATGTCC
AT1G80360-qPCR-R	ACAATGTGGTCTCCCTCAAC
GmVAS1-NdeI-F	CCCATATGATGGGTTCGTTCGTGAAG
GmVAS1-PstI-R	AACTGCAGTTACTCCCGCAGTCCGTCT
GmActin11-qPCR-F	ATCTTGACTGAGCGTGGTTATTCC
GmActin11- qPCR-R	GCTGGTCCTGGCTGTCTCC
GmVAS1-1- qPCR-F	TGATGGCCTGAAACACTCTTG
GmVAS1-1- qPCR-R	GCAAAGTCTTTTACTTCAGAGGG
GmVAS1-2- qPCR-F	CAAAGCTCCCAGACTTAGACG
GmVAS1-2- qPCR-R	TGCAGTCATTCTCCGTCAAG
pGBKT7-T7	TAATACGACTCACTATAGGGCGAGC
pGBKT7-ADR	GTGAACTTGCGGGGTTTTTCAGTAT

图1 VAS1基因家族进化分析和基因结构大豆（Glycine max）：Glyma.15G012300, Glyma.13G361500；菜豆（Phaseolus vulgaris）：Phvul.005G173300；豇豆（Vigna unguiculata）：Vigun05g288500；百脉根（Lotus corniculatus）：Lj3g0021181；苜蓿（Medicago truncatula）：Med-tr4g011820；鹰嘴豆（Cicer arietinum）：Ca13475；烟草（Nicotiana tabacum）：The.06G081300；雷蒙德氏棉（Gossypium raimondii）：Gorai.009G132000；油菜（Brassica campestris）：Brara.G02028, Brara.B02505, Brara.G03736；拟南芥（Arabidopsis thaliana）：AT1G80360；栽培黄瓜（Cucumis sativus）：Cucsa.004520

Fig. 1 Phylogenetic tree and gene structure of VAS1 gene family

图2 大豆VAS1基因家族顺式作用元件预测

Fig. 2 Prediction of cis-acting elements of GmVAS1（VAS1 in G. max）

图3 VAS1蛋白氨基酸保守基序分析

Fig. 3 Conservative motifs of VAS1 protein amino acids

图4 大豆VAS1基因家族不同种子发育时期各组织表达模式

Fig. 4 Tissue expression pattern of soybean VAS1 gene family at different embryonic stage

图5 PEG和NaCl处理下GmVAS1基因家族的表达模式 *：P<0.05；**：P<0.01。下同

Fig. 5 Expression pattern of GmVAS1 gene family under PEG or NaCl treatment *: P<0.05; **: P<0.01. The same below

图6 不同浓度硝酸盐和PEG协同处理下GmVAS1表达模式 NN：适宜浓度硝酸盐；HN：高浓度硝酸盐；LN：低浓度硝酸盐；NNPEG：适宜浓度硝酸盐和PEG复合处理；HNPEG：高浓度硝酸盐和PEG复合处理；LNPEG：低浓度硝酸盐和PEG复合处理

Fig. 6 GmVAS1 expression patterns under co-treatment of different concentrations of nitrate and PEG NN: Normal concentration nitrate. HN: High concentration nitrate. LN: Low concentration nitrate. NNPEG: Normal concentration nitrate and PEG composite treatment. HNPEG: High concentration nitrate and PEG composite treatment. LNPEG: Low concentration nitrate and PEG composite treatment

图7 拟南芥vas1突变体鉴定 A：T-DNA插入位置；B：T-DNA插入鉴定；C：RNA水平分析

Fig. 7 Mutant identification vas1 in Arabidopsis thaliana A: T-DNA insertion location in vas1. B: T-DNA insertion identification. C: RNA level identification

图8 拟南芥转基因植株鉴定 A：转基因拟南芥阳性植株筛选（M：DNA分子标准；1：正对照pMDC83-GmVAS1-1质粒）；B：转基因拟南芥中GmVAS1-1相对表达量

Fig. 8 Identification of transgenic A. thaliana plant A: Screening of transgenic A. thaliana plants（M: DNA marker. 1: Positive control pMDC83-GmVAS1-1 plasmid）. B: Relative expression of GmVAS1-1 in transgenic A. thaliana

图9 转基因拟南芥侧根发育 A：苗期根部表型；B：侧根数统计

Fig. 9 Lateral root development in transgenic A. thaliana plants A: Phenotype of roots during seedling. B: Statistic of lateral root in various plants

表2 相互作用因子

Table 2 Interaction factors

基因Gene ID	基因注释Gene description	筛选方式Screening method
Glyma.12G037400 Glyma.14G189400	果糖-二磷酸醛缩酶1/果糖-1,6-二磷酸醛缩酶 Fructose-bisphosphate aldolase 1/Fructose-1,6-bisphosphate triosephosphate-lyase	酵母双杂交试验 Yeast hybrid screening
Glyma.04G065600	双功能抑制剂/脂质转移蛋白/种子储存2s白蛋白超家族蛋白 Bifunctional inhibitor /Lipid-transfer protein / Seed storage 2s albumin super family protein
Glyma.12G207600	成束蛋白样阿拉伯半乳聚糖蛋白13相关蛋白 Fasciclin-like arabinogalactan protein 13-related
Glyma.01G063000	UDP-阿拉伯吡喃糖突变酶UDP-arabinopyranose mutase
Glyma.06G064900	与膜相关的泛素蛋白连接酶//未命名亚族Membrane associated ring finger // Subfamily not named
Glyma.08G261200 Glyma.20G148900	甲基四氢叶酸：同型半胱氨酸甲基转移酶相关/同型半胱氨酸S-甲基转移酶 Methyltetrahydrofolate: Homocysteine methyltransferase related/Homocysteine S-methyltransferase	STRING数据库在线预测 STRING database
Glyma.02G087900 Glyma.10G172700 Glyma.13G001200	蛋氨酸-γ-裂解酶Methionine-gamma-lyase
Glyma.10G244300 Glyma.20G150100 Glyma.20G150200	顺式还原酮双加氧酶/1,2-二羟基-3-酮-5-甲硫戊烯双加氧酶4 Acireductone dioxygenase / 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase 4
Glyma.15G012300	亚组I转氨酶相关//未命名亚族Subgroup I aminotransferase related // Subfamily not named
Glyma.19G158800	同型半胱氨酸S-甲基转移酶/硒代半胱氨酸Se-甲基转移酶 Homocysteine S-methyltransferase/Selenocysteine Se-methyltransferase
Glyma.03G158400	吲哚-3-甘油-磷酸裂解酶/组蛋白去乙酰化酶抑制剂/色氨酸合酶/色氨酸合成酶 Indole-3-glycerol-phosphate lyase / TSA / Tryptophan synthase / Tryptophan synthetase

表2 相互作用因子

Table 2 Interaction factors

基因Gene ID	基因注释Gene description	筛选方式Screening method
Glyma.12G037400 Glyma.14G189400	果糖-二磷酸醛缩酶1/果糖-1,6-二磷酸醛缩酶 Fructose-bisphosphate aldolase 1/Fructose-1,6-bisphosphate triosephosphate-lyase	酵母双杂交试验 Yeast hybrid screening
Glyma.04G065600	双功能抑制剂/脂质转移蛋白/种子储存2s白蛋白超家族蛋白 Bifunctional inhibitor /Lipid-transfer protein / Seed storage 2s albumin super family protein
Glyma.12G207600	成束蛋白样阿拉伯半乳聚糖蛋白13相关蛋白 Fasciclin-like arabinogalactan protein 13-related
Glyma.01G063000	UDP-阿拉伯吡喃糖突变酶UDP-arabinopyranose mutase
Glyma.06G064900	与膜相关的泛素蛋白连接酶//未命名亚族Membrane associated ring finger // Subfamily not named
Glyma.08G261200 Glyma.20G148900	甲基四氢叶酸：同型半胱氨酸甲基转移酶相关/同型半胱氨酸S-甲基转移酶 Methyltetrahydrofolate: Homocysteine methyltransferase related/Homocysteine S-methyltransferase	STRING数据库在线预测 STRING database
Glyma.02G087900 Glyma.10G172700 Glyma.13G001200	蛋氨酸-γ-裂解酶Methionine-gamma-lyase
Glyma.10G244300 Glyma.20G150100 Glyma.20G150200	顺式还原酮双加氧酶/1,2-二羟基-3-酮-5-甲硫戊烯双加氧酶4 Acireductone dioxygenase / 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase 4
Glyma.15G012300	亚组I转氨酶相关//未命名亚族Subgroup I aminotransferase related // Subfamily not named
Glyma.19G158800	同型半胱氨酸S-甲基转移酶/硒代半胱氨酸Se-甲基转移酶 Homocysteine S-methyltransferase/Selenocysteine Se-methyltransferase
Glyma.03G158400	吲哚-3-甘油-磷酸裂解酶/组蛋白去乙酰化酶抑制剂/色氨酸合酶/色氨酸合成酶 Indole-3-glycerol-phosphate lyase / TSA / Tryptophan synthase / Tryptophan synthetase

图10 酵母双杂交文库筛选获得的阳性克隆 P53/Large T：系统正对照；Lamin C/Large T：负对照

Fig. 10 Positive clones obtained from yeast two hybrid library screening P53/Large T: The positive control. Lamin C/Large T: The negative control

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