检索
E-mail
RSS
高级检索 图表检索

当期目录 我要投稿 过刊浏览
高级检索 图表检索

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

RichHTML

8

PDF (PC)

372

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

[1] 黄筱玲. 植物病毒病化学防治剂的研究现状及应用前景. 湖北化工, 1996, 13(S1):21-23.
[2] 林祥永, 陈飞雄, 王金文, 等. 烟草花叶病的发生规律与防治策略. 安徽农业科学, 2003, 31(3):487.
[3] 陈启建, 刘国坤, 吴祖建, 等. 三叶鬼针草中黄酮甙对烟草花叶病毒的抑制作用. 福建农林大学学报(自然科学版), 2003, 32(2):181-184.
[4] 颜培强, 白先权, 万秀清, 等. 应用RNAi技术培育抗TMV病毒转基因烟草. 遗传, 2007, 29(8):1018-1022.
[5] Stange C, Matus J T, Elorza A, et al. Identification and characterization of a novel tobacco mosaic virus resistance N gene homologue in Nicotiana tabacum plants. Functional Plant Biology, 2004, 31(2):149-158.
doi: 10.1071/FP03160
[6] Wang J, Wang X R, Qi Z, et al. iTRAQ protein profile analysis provides integrated insight into mechanisms of tolerance to TMV in tobacco (Nicotiana tobacum). Journal of Proteomics, 2016, 132(2):21-30.
doi: 10.1016/j.jprot.2015.11.009
[7] Bachmann M, Huber J L, Liao P C, et al. The inhibitor protein of phosphorylated nitrate reductase from spinach (Spinacia oleracea) leaves is a 14-3-3 protein. Federation of European Biochemical Societies Letters, 1996, 387(2/3):127.
doi: 10.1016/0014-5793(96)00478-4
[8] Soll T M. 14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants. The Plant Cell, 2000, 12(1):53-64.
doi: 10.1105/tpc.12.1.53
[9] Denison F C, Paul A L, Zupanska A K, et al. 14-3-3 proteins in plant physiology. Seminars in Cell and Developmental Biology, 2011, 22(7):720-727.
doi: 10.1016/j.semcdb.2011.08.006 pmid: 21907297
[10] Nina J, Christian T, Claudia O. Arabidopsis 14-3-3 proteins: fascinating and less fascinating aspects. Frontiers in Plant Science, 2011, 2(96):96.
[11] Albertus H, Boer, Paula J, et al. Plant 14-3-3 proteins as spiders in a web of phosphorylation. Protoplasm A, 2013, 250(2):425-440.
[12] Cotelle V, Leonhardt N. 14-3-3 proteins in guard cell signaling. Frontiers in Plant Science, 2016, 6:1210.
[13] Liu Q, Zhang S, Liu B. 14-3-3 proteins:macro-regulators with great potential for improving abiotic stress tolerance in plants. Biochemical and Biophysical Research Communications, 2016, 477(1):9-13.
doi: 10.1016/j.bbrc.2016.05.120
[14] Taylor K W, Kim J G, Su X B, et al. Tomato TFT1 is required for PAMP-triggered immunity and mutations that prevent T3S effector xopN from binding to TFT1 attenuate xanthomonas virulence. PLoS Pathogens, 2012, 8(6):e1002768.
doi: 10.1371/journal.ppat.1002768
[15] Konagaya K I, Matsushita Y, Kasahara M, et al. Members of 14-3-3 protein isoforms interacting with the resistance gene product N and the elicitor of tobacco mosaic virus. Journal of General Plant Pathology, 2004, 70(4):221-231.
[16] Malgorzata J R, Katarzyna K. The role of plasma membrane H+-ATPase in salinity stress of plants. Progress in Botany, 2015, 76:77-92.
[17] Viso F D, Casaretto J A, Quatrano R S. 14-3-3 proteins are components of the transcription complex of the ATEM1 promoter in Arabidopsis. Planta, 2007, 227(1):167-175.
doi: 10.1007/s00425-007-0604-1
[18] 周颖, 李冰樱, 李学宝. 14-3-3蛋白对植物发育的调控作用. 植物学报, 2012, 47(1):55-64.
doi: 10.3724/SP.J.1259.2012.00055
[1] 刘晓丽, 韩利涛, 魏楠, 申飞, 蔡一林. 玉米ZmGS5基因克隆、分子特性分析及对拟南芥的遗传转化[J]. 作物杂志, 2021, (1): 16–25
[2] 罗海斌, 蒋胜理, 黄诚梅, 曹辉庆, 邓智年, 吴凯朝, 徐林, 陆珍, 魏源文. 甘蔗ScHAK10基因克隆及表达分析[J]. 作物杂志, 2018, (4): 53–61
[3] 向鹏, 龙承波, 罗红丽. 水稻OsMED7基因的克隆及表达分析[J]. 作物杂志, 2013, (3): 21–24
[4] 孙方志, 王大庆, 刘慧民. 小黑杨APETALA1基因的克隆与花芽发育中的表达模式[J]. 作物杂志, 2012, (4): 34–40
[5] 史冬燕, 黄兴奇. 东乡野生稻STK抗病基因片段的克隆及序列分析[J]. 作物杂志, 2009, (2): 26–29
[6] 刘占领, 雷财林, 程治军, 等. 水稻稻瘟病抗性基因定位与克隆研究进展[J]. 作物杂志, 2007, (3): 16–19
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!