草莓FabHLH3基因克隆表达分析与过量和干涉表达载体的构建

doi:10.3969/j.issn.1004-1524.2017.02.07

浙江农业学报 ›› 2017, Vol. 29 ›› Issue (2): 220-227.DOI: 10.3969/j.issn.1004-1524.2017.02.07

草莓FabHLH3基因克隆表达分析与过量和干涉表达载体的构建

张青¹, 苗立祥², 张豫超², 杨肖芳², 蒋桂华^{2, *}

1.临安市农林技术推广中心,浙江临安 311300;
2.浙江省农业科学院园艺研究所,浙江杭州 310021

收稿日期:2016-10-14 出版日期:2017-02-15 发布日期:2017-03-06
通讯作者: 蒋桂华,E-mail:jgh2004267@sina.com
作者简介:张青(1962—),男,浙江临安人,高级农艺师,主要从事果树栽培技术推广。E-mail:zq373@163.com
基金资助:
国家自然科学基金(31201613); 浙江省自然科学基金(LY16C150004)

Cloning and expression analysis of FabHLH3 gene from cultivated strawberry and construction of overexpression and silencing vector

ZHANG Qing¹, MIAO Lixiang², ZHANG Yuchao², YANG Xiaofang², JIANG Guihua^{2, *}

1.Agricultural Technology Extension Centre of Lin'an, Lin'an 311300, China;;
2.Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2016-10-14 Online:2017-02-15 Published:2017-03-06

摘要/Abstract

摘要： 以八倍体草莓品种越心(Fragaria×ananassa cv. Yuexin)为试材,利用RT-PCR技术从果实中克隆出FabHLH3基因全长编码序列,运用生物学软件对该序列进行相关生物信息学分析,同时采用实时荧光定量PCR方法对FabHLH3基因在不同组织、果实不同发育阶段及不同光质处理下的表达模式进行研究。测序和分析结果表明,FabHLH3基因的CDS全长2 094 bp,编码697个氨基酸残基,该蛋白质理论分子量约为77.43 ku、等电点(pI)为6.05;经DNAMAN分析,核苷酸序列和氨基酸序列与二倍体森林草莓(F. vesca)FvbHLH3基因的一致性分别为92.04%和90.87%。利用实时荧光定量PCR检测FabHLH3基因在叶、叶柄、花和果实中的表达情况,发现在叶中的表达量最高,随着果实发育,FabHLH3基因表达量逐渐增加,在半红期达到最高,红果期开始下降。此外,FabHLH3基因的表达还受到红光和蓝光的诱导。用无缝克隆技术构建了FabHLH3基因的过量表达载体pGreen-FabHLH3和干涉表达载体pGreen-FabHLH3i,为进一步验证FabHLH3基因的功能奠定了基础。

关键词: 草莓, FabHLH3, 基因克隆, 表达模式, 过表达和干涉表达载体

Abstract: The full-length cDNA sequence of FabHLH3 gene was cloned from strawberry (Fragaria×ananassa cv. Yuexin) through homology cloning method. Then, bioinformatics and expression pattern of FabHLH3 gene were analyzed by some softwares and real-time fluorescent quantitative PCR technique, respectively. Sequence analysis showed that the cDNA of FabHLH3 was 2 094 bp, encoding 697 amino acids, while the estimated molecular weight and isoelectric point of the putative protein were 77.43 ku and 6.05. After DNAMAN analysis, it shared 92.04% and 90.87% identity with the nucleotide sequence and amino acid sequence of bHLH3 gene from woodland strawberry(F. vesca), respectively. The expression of FabHLH3 was analyzed in leaf, petiole, flower and fruit being grown under colored light-quality plastic film. The results showed that FabHLH3 was expressed in all the tissues and the highest level was found in leaf. The expression analysis showed that the expression level of FabHLH3 was gradually increased during fruit development. The lowest expression was detected at small green fruit stage, and a reduced peak appeared at the full red stage. In addition, the expression of FabHLH3 gene can be induced by red and blue light. The overexpression and silencing vectors of FabHLH3, pGreen-FabHLH3 and pGreen-FabHLH3i, were constructed by in-fusion method with a plant expression vector pGreenII 62-SK. It would be used in functional analysis in the future research.

Key words: strawberry, FabHLH3, gene cloning, expression pattern, overexpression and silencing vector

中图分类号:

S668.4

张青, 苗立祥, 张豫超, 杨肖芳, 蒋桂华. 草莓FabHLH3基因克隆表达分析与过量和干涉表达载体的构建[J]. 浙江农业学报, 2017, 29(2): 220-227.

ZHANG Qing, MIAO Lixiang, ZHANG Yuchao, YANG Xiaofang, JIANG Guihua. Cloning and expression analysis of FabHLH3 gene from cultivated strawberry and construction of overexpression and silencing vector[J]. , 2017, 29(2): 220-227.

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草莓FabHLH3基因克隆表达分析与过量和干涉表达载体的构建

Cloning and expression analysis of FabHLH3 gene from cultivated strawberry and construction of overexpression and silencing vector

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