马铃薯扩展蛋白基因<i>StEXLB1a</i>的表达模式分析及功能初步鉴定

doi:10.7668/hbnxb.20195691

摘要/Abstract

摘要： 探究马铃薯基因StEXLB1a的表达模式和功能,为后续马铃薯病害的抗性研究提供理论依据。在东北农业大学植物资源与分子生物学实验室前期研究基础上,以马铃薯大西洋品种为材料,克隆了StEXLB1a基因并对其进行了生物信息学、表达模式和抗病功能初步分析。结果表明:StEXLB1a基因cDNA全长序列768 bp,编码255个氨基酸,注释显示其属于扩展蛋白家族基因。亚细胞定位显示,该蛋白定位于细胞壁上。在马铃薯不同组织中,StEXLB1a基因在叶中表达量最高,根和地上茎其次,在花和块茎中最低。在激素(吲哚乙酸、赤霉素、脱落酸)、逆境(高温、低温、盐、干旱)和真菌病害干腐病(燕麦镰孢菌)、细菌病害软腐病(胡萝卜软腐欧文氏菌)及青枯病(茄科雷尔氏菌)等多种胁迫处理下,StEXLB1a基因的表达量在各个处理中均有显著变化。获得过表达转基因马铃薯植株后,接种干腐病致病菌燕麦镰孢菌,转基因比野生型植株叶片受到病菌的侵染严重。过表达植株中的活性氧清除系统相关酶活性发生显著变化,POD、SOD活性受到抑制,MDA含量高于野生型植株,植株受损程度加重。分析结果表明,过表达StEXLB1a的转基因马铃薯植株相较野生型对干腐病的抗病性下降。

关键词: 马铃薯, 扩展蛋白, 基因克隆, 表达模式分析, 功能鉴定

Abstract:

To explore the expression pattern of potato gene StEXLB1a and provide a theoretical basis for the resistance to potato disease,based on the previous research of the Laboratory of Plant Resources and Molecular Biology at Northeast Agricultural University,the study using the Atlantic variety of potato as material, cloned the StEXLB1a gene, and preliminarily analyzed its bioinformatics, expression pattern, and disease resistance. The results showed that the full-length cDNA sequence of StEXLB1a gene was 768 bp,encoding 255 amino acids.The annotation indicated that it belonged to the extend protein family genes.Subcellular localization showed that the protein was localized to the cell wall.Among different tissues of potato,StEXLB1a gene expression was highest in leaves,followed by roots and stems,and lowest in flowers and tubers.The expression level of StEXLB1a gene changed significantly under various stress treatments,such as hormone(indoleacetic acid,gibberellin,abscisic acid),stress(high temperature,low temperature,salt,drought),fungal disease dry rot(Fusarium avenaceum),bacterial disease soft rot(Erwinia carotovora subsp,Ecc)and bacterial wilt(Ralstonia solanacearum,RS).The overexpressed transgenic potato plants were inoculated with the dry rot pathogen Fusarium avenaceum,and the transgenic plants were more seriously infected than the wild-type plants.The enzyme activities of active oxygen scavenging system in overexpressed plants were significantly changed,POD and SOD activities were inhibited,MDA content was higher than that of wild-type plants,and the damage degree of plants was aggravated.The results showed that transgenic potato plants with overexpression of StEXLB1a had lower resistance to dry rot than those with wild type.

Key words: Potato, Expansin, Gene cloning, Expression pattern analysis, Functional identification

中图分类号:

杜冲, 贺付蒙, 隋嘉, 赵潇璨, 车云竹, 张增利, 刘丹, 王雪, 李凤兰. 马铃薯扩展蛋白基因StEXLB1a的表达模式分析及功能初步鉴定[J]. 华北农学报, 2025, 40(5): 161-170. doi: 10.7668/hbnxb.20195691.

DU Chong, HE Fumeng, SUI Jia, ZHAO Xiaocan, CHE Yunzhu, ZHANG Zengli, LIU Dan, WANG Xue, LI Fenglan. Expression Pattern Analysis and Functional Identification of Potato Expansion Protein Gene StEXLB1a[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(5): 161-170. doi: 10.7668/hbnxb.20195691.

http://www.hbnxb.net/CN/Y2025/V40/I5/161

图/表 12

表1 StEXLB1基因引物序列

Tab.1 Primer sequence of StEXLB1

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	功能 Function
StEXLB1a-F	ATGGCTCCTCTTCAAGTTCT	克隆基因
StEXLB1a-R	TTATGCATCTTCTTGTATTC
Q-StEXLB1a-F	ACTGTAGTTGTAACAGACCAAGG	表达特异性分析
Q-StEXLB1a-R	GTGGCAATATAGAAACAGCAGCA
Q-β-actin-F	GCTTCCCGATGGTCAAGTCA
Q-β-actin-R	GGATTCCAGCTGCTTCCATTC
LB1a-PstⅠ-F	AAAA CTGCAGATGGCTCCTCTTCAAGTTCTCTCTG	亚细胞定位载体构建
LB1a-SalⅠ-R	ACGC GTCGACTGCATCTTCTTGTATTCCTGAGTCG
LB1a-XbaⅠ-F	GC TCTAGAATGGCTCCTCTTCAAGTTCT	过表达载体的构建
LB1a-BamHⅠ-R	CG GGATCCTTATGCATCTTCTTGTATTC
GUS-F	GTCGCGCAAGACTGTAACCA	过表达植株鉴定
GUS-R	CGGCGAAATTCCATACCTG

图1 PCR扩增产物电泳结果 M.DL2000 Marker;1,2.目的片段。

Fig.1 Electrophoresis results of PCR amplification products M.DL2000 Marker;1,2.Target fragment.

图2 StEXLB1a蛋白的生物信息学分析 A.StEXLB1a蛋白保守结构域分析;B.StEXLB1a蛋白信号肽预测;C.StEXLB1a蛋白亲水疏水性预测;D.StEXLB1a蛋白二级结构预测;E.StEXLB1a蛋白三级结构预测。

Fig.2 Bioinformatics analysis of StEXLB1a protein A.Conserved domain analysis of StEXLB1a protein;B.StEXLB1a protein signal peptide prediction;C.Prediction of hydrophilic and hydrophobic properties of StEXLB1a protein;D.StEXLB1a protein secondary structure prediction;E.Prediction of the tertiary structure of StEXLB1a protein.

图3 StEXLB1a蛋白的系统进化发育树

Fig.3 Phylogenetic tree of StEXLB1a protein

图4 马铃薯不同组织中StEXLB1a的相对表达量不同的字母表示存在显著性差异(P<0.05)。图5—7,9,11同。

Fig.4 Relative expression of StEXLB1a in different potato tissues Different letter indicate significant differences(P<0.05). The same as Fig.5—7,9,11.

图5 不同时间下外源激素(IAA、GA₃、ABA)对马铃薯叶片StEXLB1a相对表达量的影响

Fig.5 Effects of exogenous hormones(IAA,GA₃,ABA)on the relative expression of StEXLB1a in potato leaves at different time

图6 不同时间下非生物胁迫(高温、低温、NaCl、PEG)对马铃薯叶片StEXLB1a相对表达量的影响

Fig.6 Effects of abiotic stress(high temperature,low temperature,NaCl,PEG) on the relative expression of StEXLB1a in potato leaves at different time

图7 StEXLB1a基因的抗病表达特异性分析

Fig.7 Analysis of resistance specificity of StEXLB1a gene

图8 StEXLB1a蛋白的亚细胞定位

Fig.8 Subcellular localization of StEXLB1a protein

图9 马铃薯试管薯的遗传转化、阳性鉴定及过表达植株qRT-PCR检测结果 A—D.微型薯薄片生芽、分化、生根、生长;E:M.DNA分子质量标准2000,+.阳性对照(重组质粒为模板的PCR产物),-.阴性对照(野生型植株DNA为模板),1—6.转StEXLB1a基因植株DNA为模板;F.过表达植株qRT-PCR检测结果。

Fig.9 Genetic transformation,positive identification and qRT-PCR results of overexpressed potato in the potato test tube A—D.Microscopic potato slices sprouting,differentiating,rooting and growing;E:M.DNA molecular weight standard 2000,+.Positive control(PCR product with recombinant plasmid as template),-.Negative control(DNA from wild-type plants as template),1—6.DNA from transgenic StEXLB1a plants as template;F.qRT-PCR detection results of overexpression plants.

图10 致病菌处理后叶片病斑变化

Fig.10 Changes of leaf lesions after pathogen treatment

图11 致病菌处理转基因植株叶片后抗病相关生理指标测定

Fig.11 Determination of physiological indexes of disease resistance in transgenic plant leaves treated with pathogenic bacteria

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马铃薯扩展蛋白基因StEXLB1a的表达模式分析及功能初步鉴定

Expression Pattern Analysis and Functional Identification of Potato Expansion Protein Gene StEXLB1a

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马铃薯扩展蛋白基因StEXLB1a的表达模式分析及功能初步鉴定

Expression Pattern Analysis and Functional Identification of Potato Expansion Protein Gene StEXLB1a

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