小麦与叶锈病菌互作过程中转录因子基因TaNAC35的功能分析  

Functional Analysis of Transcription Factor Gene TaNAC35 in the Interaction Between Wheat(Triticum aestivum) and Puccinia triticina

作　　者：赵晨光 张娜[1,2] 温晓蕾 杨文香[1,2] 张娜[1,2] 刘大群 ZHAO Chen-Guang;ZHANG Na;WEN Xiao-Lei;YANG Wen-Xiang;ZHANG Na;LIU Da-Qun(College of Plant Protection,Hebei Agricultural University,Baoding 071000,China;Technological Innovation Center for Biological Control of Crop Diseases and Insect Pests of Hebei Province,Baoding 071000,China;Hebei Normal University of Science&Technology,Qinhuangdao 066000,China)

机构地区：[1]河北农业大学植物保护学院,保定071000 [2]河北省农作物病虫害生物防治技术创新中心,保定071000 [3]河北科技师范学院,秦皇岛066000

出　　处：《农业生物技术学报》2022年第1期15-24,共10页Journal of Agricultural Biotechnology

基　　金：河北省自然科学基金(C2021204055);国家自然科学基金(31571956);河北省高等学校科学技术研究项目(QN2018148)。

摘　　要：NAC (NAM-ATAF1/2-CUC2)蛋白是植物特有的一种转录因子,在植物生长发育和胁迫应答中都具有重要作用。本课题组前期在小麦(Triticum aestivum)与叶锈菌(Puccinia triticina)互作的转录组数据库中进行了差异基因筛选,在小麦近等基因系TcLr3ka中克隆了显著差异表达的TaNAC35基因全长。本研究通过同源扩增分离TaNAC35在TcLr3ka中的基因序列,利用MEGA软件将基因组序列与CDS序列比对,发现其编码区具有4个外显子和3个内含子;启动子元件预测结果显示,TaNAC35启动子区域有多种激素响应元件、光响应元件及转录因子结合位点。进一步构建重组质粒pGR107-TaNAC35-GFP并瞬时转化烟草(Nicotiana tabacum)叶片,荧光显微镜观察发现TaNAC35蛋白定位于细胞核与细胞膜中。qPCR检测发现,TaNCA35基因表达具有组织特异性,在小麦叶片中的表达量最高,并参与小麦对叶锈菌胁迫及脱落酸和水杨酸刺激的响应。利用病毒诱导的基因沉默(virus induced gene silencing, VIGS)技术发现,沉默TaNAC35的植株对叶锈病菌的侵染具有抑制作用。上述结果表明,TaNAC35在小麦抗叶锈病菌过程中起负调控作用。本研究为深入探讨NAC转录因子在小麦响应叶锈病菌胁迫中的作用提供了参考依据。NAC(NAM-ATAF1/2-CUC2) proteins are plant specific transcription factors, and play important roles in regulation of growth, development and stress response in plant. In the previous studies, TaNAC35 was cloned from wheat(Triticum aestivum) near iso-geneic line TcLr3ka based on the database of transcriptome RNA-seq from wheat and Puccinia triticina(Pt) interaction, in which the expression of TaNAC35 was significantly different between inoculated and uninoculated wheat. This study isolated the genome DNA(gDNA) sequence of TaNAC35 in TcLr3ka, and compared with CDS sequence by MEGA software. The results showed 4 exons and 3 introns in the gDNA sequence of TaNAC35. The promoter elements were predicted and analyzed, and the results showed that there were various of hormone responsive elements, light responsive elements and transcription factor binding sites. Recombinant plasmid pGR107-TaNAC35-GFP was constructed and transiently transformed into tobacco(Nicotiana tabacum), which revealed that TaNAC35 localized in the nucleus and the cell membrane. qPCR analysis showed TaNCA35 was highly expressed in leaves and might be involved in the response of wheat to Pt, abscisic acid and salicylic acid. Virus-induced gene silencing(VIGS)was employed to silence part of TaNAC35 sequence, the results showed that compared with TaNAC35 nonsilenced wheat(control), the growth and development of Pt on TaNAC35 silenced wheat decreased significantly,which could inhibit the infection of the pathogen. The results in this study showed that TaNAC35 played a negative regulatory role in the resistance of wheat to Pt, which provides a reference for further exploration of the role of NAC transcription factors in the interaction between wheat and Pt.

关 键 词：小麦叶锈病 NAC(NAM-ATAF1/2-CUC2)转录因子 表达分析 病毒诱导的基因沉默(VIGS) 

分 类 号：S435[农业科学—农业昆虫与害虫防治]