马铃薯StRac5、StRac7和StRac13基因的克隆及其表达模式分析

doi:10.7668/hbnxb.20194582

摘要/Abstract

摘要：

探究马铃薯基因的表达模式,为后续研究基因对马铃薯病害的抗性影响提供理论依据。以马铃薯为试验材料,克隆获得3个马铃薯内源小G蛋白基因StRac5、StRac7、StRac13,并对其进行生物信息学分析和表达模式分析。结果表明,StRac5、StRac7、StRac13与拟南芥和水稻中的小G蛋白含有相似的保守序列,均属于ROP蛋白。StRac5和StRac13的蛋白结构域与拟南芥AtRop1、AtRop3、AtRop5、AtRop6及水稻OsRac5、OsRac6、OsRac7相同,StRac7与拟南芥AtRop9的蛋白结构域相同。系统进化树分析表明,StRac7属于第Ⅱ类,与拟南芥AtRop9遗传距离最近;StRac13属于第Ⅲ类,与拟南芥AtRop7遗传距离最近;StRac5属于第Ⅳ类,与水稻OsRac5和OsRac6遗传距离最近。在马铃薯不同组织中,StRac5、StRac7、StRac13基因的相对表达量均为叶>根>茎;与其在茎中表达量比较,StRac5、StRac7和StRac13在叶中的表达量分别增加了1 222.4%,2 531.3%,468.2%;接种晚疫菌后,StRac5、StRac7和StRac13基因的相对表达量均出现先升高后降低的趋势,StRac5和StRac13基因的相对表达量在接菌24 h后较0 h分别上调了62.2%,40.4%,StRac7基因的相对表达量在接菌72 h后较0 h上调了827.8%;经过ABA、SA、GA和6-BA处理后,StRac5、StRac7、StRac13基因的相对表达量呈现下调的趋势;JA和IAA处理后,StRac5、StRac7、StRac13基因的相对表达量呈现上调的趋势。因此,StRac5、StRac7和StRac13基因能够响应马铃薯晚疫病的侵染,在不同组织和不同激素处理下表达模式也存在一定差异。

关键词: 马铃薯, 小G蛋白, 基因克隆, 晚疫病, 表达模式

Abstract:

To explore the expression patterns of potato genes and provide theoretical basis for the subsequent study of gene resistance to potato diseases.Three endogenous small G protein genes StRac5,StRac7 and StRac13 were cloned from potato,and their bioinformatics and expression patterns were analyzed.The results showed that StRac5,StRac7 and StRac13 contained similar conserved sequences to the small G proteins in Arabidopsis and rice,all belonging to ROP proteins.The protein domains of StRac5 and StRac13 were identical with AtRop1,AtRop3,AtRop5,AtRop6 of Arabidopsis and rice OsRac5,OsRac6,OsRac7,and StRac7 was identical with AtRop9.Phylogenetic tree analysis showed that StRac7 belonged to class Ⅱ and was closest to Arabidopsis AtRop9.StRac13 belonged to class Ⅲ and was the closest genetic relative to Arabidopsis AtRop7.StRac5 belonged to class IV and was the closest genetic relative to rice OsRac5 and OsRac6.The relative expression levels of StRac5,StRac7 and StRac13 genes in different tissues of potato were leaf>root>stem.Compared with the expression in stems,the expression of StRac5,StRac7 and StRac13 in leaves were increased by 1 222.4%,2 531.3% and 468.2%,respectively.After inoculation,the relative expression of StRac5,StRac7 and StRac13 genes increased first and then decreased,and the relative expression of StRac5 and StRac13 genes increased by 62.2%,40.4%,respectively,compared with 0 h after inoculation 24 h.The relative expression of StRac7 gene was increased by 827.8% at 72 h after inoculation compared with 0 h.After ABA,SA,GA and 6-BA treatment,the relative expression of StRac5,StRac7 and StRac13 genes showed a downward trend.After JA and IAA treatment,the relative expression of StRac5,StRac7 and StRac13 genes showed an up-regulated trend.Therefore,StRac5,StRac7 and StRac13 genes can respond to potato late blight infection,and their expression patterns are different in different tissues and under different hormone treatments.

Key words: Potato, Small G protein, Gene cloning, Late blight, Expression pattern

高婉婷, 刘志达, 孙学桃, 李志平, 吕文霞, 李爱珍, 赵君, 张之为. 马铃薯StRac5、StRac7和StRac13基因的克隆及其表达模式分析[J]. 华北农学报, 2024, 39(5): 33-41. doi: 10.7668/hbnxb.20194582.

GAO Wanting, LIU Zhida, SUN Xuetao, LI Zhiping, LÜ Wenxia, LI Aizhen, ZHAO Jun, ZHANG Zhiwei. Cloning and Expression Patterns of StRac5,StRac7 and StRac13 Genes in Potato[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(5): 33-41. doi: 10.7668/hbnxb.20194582.

http://www.hbnxb.net/CN/Y2024/V39/I5/33

图/表 11

表1 引物序列信息

Tab.1 Information of primer sequences

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	引物用途 Purpose of primer
StRac5-F	ATGAGTGCTTCTAGGTTTATAAAGTG	基因克隆
StRac5-R	TCACAAGATAGAGCAGGCTTTC
StRac7-F	ATGAATGCTTCAAAGTTCATCAAATG
StRac7-R	CTAAGCAACACAGCCTCCACA
StRac13-F	ATGACTACTGCTAGATTCATCAAATG
StRac13-R	TCAAAGTATTGCACATAACGTCCTT
qStActin-F	CACCCTGTTCTGCTCACT	表达量分析
qStActin-R	CAGCCTGAATAGCAACATAC
qStRac5-F	GGACCATCCAGGTGCTGTTC
qStRac5-R	GCGGCATCAAAAACAGCCTTA
qStRac7-F	TTCAGTGCCAATGTAGCCGT
qStRac7-R	CGACGAAGTTCAGGCATCCA
qStRac13-F	ATGTGGTGGTGGATGGTAGC
qStRac13-R	TGTTCTCATAGCTTGCCTTGCT

图1 StRac5、StRac7和StRac13基因的克隆

Fig.1 Cloning of StRac5,StRac7,and StRac13 genes M.DM2000 DNA Marker。

图2 StRac5基因的cDNA序列及其编码的氨基酸序列

Fig.2 cDNA sequence of StRac5 gene and its encoded amino acid sequence

图3 StRac7基因的cDNA序列及其编码的氨基酸序列

Fig.3 cDNA sequence of StRac7 gene and its encoded amino acid sequence

图4 StRac13基因的cDNA序列及其编码的氨基酸序列

Fig.4 cDNA sequence of StRac13 gene and its encoded amino acid sequence

图5 StRac5、StRac7和StRac13蛋白保守序列比较

Fig.5 Comparison of conserved sequences of StRac5,StRac7 and StRac13 proteins

图6 马铃薯StRac5、StRac7和StRac13蛋白保守结构域(Motif)分布

Fig.6 Distribution of protein conserved domains(Motifs) of StRac5,StRac7 and StRac13 in potato

图7 马铃薯StRac5、StRac7和StRac13蛋白系统进化树

Fig.7 Phylogenetic tree of StRac5,StRac7 and StRac13 proteins in potato

图8 StRac5、StRac7和StRac13基因在马铃薯不同组织中的相对表达量不同小写字母表示不同处理间差异显著(P<0.05)。图9-10同。

Fig.8 Relative expression of StRac5,StRac7 and StRac13 genes in different tissues of potato Different lowercase letters represent significant difference in different treatment(P<0.05).The same as Fig.9-10.

图9 接晚疫菌后StRac5、StRac7和StRac13基因的相对表达量

Fig.9 Relative expression of StRac5,StRac7 and StRac13 genes after inoculation with Phytophthora latella

图10 外源激素处理后StRac5、StRac7和StRac13基因的相对表达量

Fig.10 Relative expression of StRac5,StRac7 and StRac13 genes after exogenous hormone treatment

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