甘蔗分子伴侣ShDnaJ基因克隆及在甘蔗线条花叶病毒胁迫下的表达分析  

Cloning of molecular chaperone ShDnaJ gene in sugarcane and its expression analysis under stress of sugarcane streak mosaic virus

作　　者：冯小艳[1] 王文治[1] 王俊刚[1] 沈林波[1] 赵婷婷[1] 冯翠莲[1] 张树珍[1] FENG Xiao-yan;WANG Wen-zhi;WANG Jun-gang;SHEN Lin-bo;ZHAO Ting-ting;FENG Cui-lian;ZHANG Shu-zhen(Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciences,Haikou,Hainan 571101,China)

机构地区：[1]中国热带农业科学院热带生物技术研究所,海南海口571101

出　　处：《南方农业学报》2023年第8期2310-2319,共10页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(32001604);海南省自然科学基金项目(320QN333)。

摘　　要：【目的】克隆甘蔗分子伴侣基因Sh DnaJ,并分析其在甘蔗线条花叶病毒(SCSMV)胁迫下的表达模式,为探究ShDnaJ基因在甘蔗应答SCSMV侵染过程中的功能和作用机制提供理论参考。【方法】通过同源克隆技术克隆ShDnaJ基因完整开放阅读框(ORF)序列,利用生物信息学软件对其进行分析,再利用绿色荧光蛋白(GFP)融合表达法分析ShDnaJ蛋白的亚细胞定位,采用实时荧光定量PCR检测ShDnaJ基因在甘蔗不同组织及在SCSMV胁迫下的表达水平。【结果】ShDnaJ基因ORF全长1260 bp,编码419个氨基酸残基,蛋白分子量为45682.47 Da,理论等电点(pI)为8.78,属于稳定的亲水蛋白,含有DnaJ结构域,属于DnaJ超家族成员,定位于内质网。ShDnaJ蛋白的二级结构由113个α-螺旋、31个β-转角、78个延伸链和197个无规则卷曲组成。ShDnaJ蛋白与高粱SbDnaJ蛋白的氨基酸序列相似性最高,为96.93%,说明两者亲缘关系最近。ShDnaJ基因在甘蔗根、茎和叶中均有表达,且根和茎中的相对表达量显著高于叶中(P<0.05,下同),但未成熟茎Sh DnaJ基因的相对表达量高于成熟茎;成熟叶ShDnaJ基因的相对表达量高于未成熟叶。随着处理时间的推移,SCSMV胁迫组和对照组(磷酸缓冲液处理)中ShDnaJ基因的相对表达量整体呈下降趋势,但SCSMV接种后不同时间点ShDnaJ基因的相对表达量均显著高于对照组。【结论】ShDnaJ基因具有组织表达特异性,且组织的成熟程度会影响其表达水平。SCSMV胁迫下该基因表达上调,推测该基因参与调控甘蔗植株对SCSMV的抗性。【Objective】The purpose of the study was to clone the molecular chaperone gene ShDnaJ in sugarcane,and analyze its expression pattern under the stress of sugarcane streak mosaic virus(SCSMV),so as to provide a theoretical reference for exploring the function and mechanism of action of ShDnaJ gene in sugarcane in response to SCSMV infection.【Method】ShDnaJ gene complete open reading frame(ORF)sequence was cloned by homologous cloning technology and was analyzed by bioinformatics software.The subcellular localization of ShDnaJ protein was analyzed by fusion expression with green fluorescent protein(GFP).The expression levels of ShDnaJ gene in different tissues of sugarcane and under SCSMV stress were detected by real-time fluorescence quantitative PCR.【Result】The open reading frame(ORF)of ShDnaJ gene was1260 bp in length,and encoded 419 amino acid residues.ShDnaJ protein,with molecular weight of 45682.47 Da and theoretical isoelectric point(pI)of 8.78,was a stable hydrophilic protein,which contained a DnaJ structural domain and belonged to the member of DnaJ superfamily.ShDnaJ protein was located in the endoplasmic reticulum.The secondary structure of ShDnaJ protein consisted of 113α-helixes,31β-turns,78 extended strands and 197 random coils.The amino acid sequence of ShDnaJ protein had the highest amino acid sequence similarity with that of SbDnaJ in sorghum,which was 96.93%,indicating that the two proteins were most closely related.ShDnaJ gene was expressed in roots,stems and leaves of sugarcane,and the expression levels in roots and stems were significantly higher than that in leaves(P<0.05,the same below).However,the expression level of ShDnaJ gene in immature stems was higher than that in mature stems.The expression level of ShDnaJ gene in mature leaves was higher than that in immature leaves.With the pass of treatment time,the relative expression of ShDnaJ gene in the SCSMV stress group and the control group(treated with phosphate buffer)showed an overall downward trend,but the expression levels o

关 键 词：甘蔗 分子伴侣DnaJ 基因克隆 甘蔗线条花叶病毒(SCSMV) 表达分析 

分 类 号：S566.103.53[农业科学—作物学]