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作物杂志,2021, 第5期: 6–13 doi: 10.16035/j.issn.1001-7283.2021.05.002

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

烟草Nt14-3-3-like C基因的克隆与功能分析

宋坤锋1(), 郝风声2, 姚欣1, 王静1, 刘卫群1()   

  1. 1河南农业大学生命科学学院,450002,河南郑州
    2中国农业大学植物保护学院,100089,北京
  • 收稿日期:2021-04-02 修回日期:2021-05-06 出版日期:2021-10-15 发布日期:2021-10-14
  • 通讯作者: 刘卫群
  • 作者简介:宋坤锋,从事烟草遗传育种研究,E-mail： laurnin@foxmail.com
  • 基金资助:
    河南省自然科学基金“14-3-3h-1蛋白与ARF1互作调控TMV侵染耐受性的分子机理”(212300410159)

Cloning and Functional Analysis of Tobacco Nt14-3-3-like C Gene

Song Kunfeng1(), Hao Fengsheng2, Yao Xin1, Wang Jing1, Liu Weiqun1()   

  1. 1College of Life Sciences, Henan Agricultural University, Zhengzhou 450002, Henan, China
    2College of Plant Protection, China Agricultural University, Beijing 100089, China
  • Received:2021-04-02 Revised:2021-05-06 Online:2021-10-15 Published:2021-10-14
  • Contact: Liu Weiqun

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摘要：

烟草花叶病毒（tobacco mosaic virus,TMV）寄主范围广泛、传染性极强,严重影响作物的产量和品质。实验室前期在不同耐受 TMV侵染的烟草品种中筛选出1个表达显著差异的蛋白14-3-3-like C,在此基础上,对Nt14-3-3-like C基因进行了克隆、生物信息学分析和亚细胞定位;通过农杆菌介导遗传转化,获取转基因植株;通过qPCR对转基因植株中TMV侵染响应相关基因的表达进行分析。结果显示,14-3-3-like C蛋白序列长度为780bp,编码260个氨基酸,具有14-3-3蛋白的典型沟槽结构,但没有跨膜结构和信号肽;定位于细胞核和细胞膜;转基因材料的qPCR分析发现,在Nt14-3-3-like C基因抑制的植株中,病原防卫和光合途径相关的基因表达显著下降,表明Nt14-3-3-like C可能通过抑制病原防卫和光合途径相关基因表达参与TMV对烟草的侵染。

关键词: TMV, 14-3-3蛋白, 病原防卫, 基因克隆

Abstract:

Tobacco mosaic virus (TMV) has a wide range of hosts and is highly infectious. TMV damages the yield and quality of crops. A protein 14-3-3-like C was screened as a candidate gene from different TMV resistant tobacco varieties. In this study, Nt14-3-3-like C was cloned and analyzed, transgenic plants were obtained by Agrobacterium-mediated transformation, qPCR was used to analyze the gene expression of transgenic plants. The results showed that Nt14-3-3-like C was 780bp in length encoding 260 amino acids, which had the typical groove structure of 14-3-3 protein, but had no transmembrane structure and signal peptide; 14-3-3-like C protein was localized in the nucleus and cell membrane. qPCR analysis of transgenic materials showed that the expression of genes related to pathogen defense and photosynthetic pathway decreased significantly in plants inhibited by Nt14-3-3-like C, these results suggested that Nt14-3-3-like C may participated in TMV infection through pathogen defense and photosynthetic pathways.

Key words: TMV, 14-3-3 protein, Pathogen defense, Gene cloning

图1

Nt14-3-3-like C PCR产物电泳图 M：DL2000 DNA标记,下同;1~3：PCR产物

图2

测序序列比对结果 Nt14-3-3-like C：克隆得到的序列;14-3-3-like C：烟草基因组数据库中的序列

图3

14-3-3-like C蛋白的生物信息学分析 a：跨膜结构预测;b：信号肽预测;c：磷酸化位点预测;d：蛋白三级结构预测

图4

14-3-3-like C同源蛋白的系统进化树分析

图5

融合GFP的35S::14-3-3-like C重组表达载体菌液PCR产物电泳 1~3：PCR产物;“-”为阴性对照

图6

14-3-3-like C蛋白亚细胞定位 绿色荧光为14-3-3-like C-GFP,红色荧光为ARR-mcherry（ARR为核标记蛋白）

图7

Nt14-3-3-like C抑制表达载体的鉴定 1~3：菌液PCR扩增产物（a）;1~2：重组载体酶切产物（b）

图8

农杆菌介导的遗传转化

图9

转基因植株PCR检测结果 1~6：PCR扩增产物的琼脂糖凝胶电泳

图10

转录水平上基因表达抑制的检测 “**”表示在0.01水平差异显著,下同。CK：对照株系;L1~L3：RNAi株系

图11

病原防卫和光合途径相关基因表达分析

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