暹罗炭疽菌转录因子CsAtf1互作蛋白的筛选和鉴定  

Identification and Screening of Proteins Interacting with CsAtf1 Transcription Factor in Colletotrichum siamense

作　　者：宋苗 方思齐 李潇[1] 刘文波[1] 林春花[1] 缪卫国[1] SONG Miao;FANG Siqi;LI Xiao;LIU Wenbo;LIN Chunhua;MIAO Weiguo(College of Plant Protection,Hainan University/Key Laboratory of Green Forest Control and Prevention,Ministry of Education Haikou,Hainan 570228,China)

机构地区：[1]海南大学植物保护学院/热带农林生物灾害绿色防控教育部重点实验室,海南海口570228

出　　处：《热带作物学报》2022年第1期60-66,共7页Chinese Journal of Tropical Crops

基　　金：海南省自然科学基金项目(No.320RC477);国家自然科学基金项目(No.31760499);现代农业产业技术体系建设专项(No.CARS-33-BC1)。

摘　　要：碱性亮氨酸拉链bZIP是真核生物转录因子家族之一,通过调控基因的表达来参与调控生长发育及生物、非生物胁迫应答等生理过程。已有报道表明bZIP转录因子Atf1与多种病原真菌的生长发育及致病力相关。由于转录因子通常能够在其他互作蛋白的参与下与顺式作用元件特异性结合,从而调节靶基因表达,因此筛选其互作蛋白对深入了解转录因子的调控机制有重要意义。本研究克隆获得来自橡胶树炭疽病的暹罗炭疽菌(Colletotrichumsiamense)的一个bZIP转录因子CsAtf1,并对CsAtf1互作蛋白进行了筛选和鉴定。研究结果显示,暹罗炭疽菌CsAtf1的DNA大小为1758 bp,cDNA为1611 bp,编码536个氨基酸,包含2个内含子,具有3个Aft1结构域和1个BRLZ结构域;利用酵母双杂技术,以pGBKT7-CsAtf1为诱饵,从暹罗炭疽菌cDNA酵母文库中筛选获得12个候选互作蛋白,包括细胞壁蛋白PhiA、Rodlet蛋白、乙醇脱氢酶、线粒体缺氧反应区蛋白、发育调控的MAPK相互作用蛋白、自噬相关蛋白、葡萄糖-甲醇-胆碱氧化还原酶、β-葡萄糖苷酶、c-4甲基固醇氧化酶、甲基转移酶和2个假定蛋白;研究还进一步利用免疫共沉淀技术证实了转录因子CsAtf1能够与线粒体缺氧反应区蛋白CsHIG发生体内互作。The basic leucine zipper(bZIP) is one of the eukaryotic transcription factor families. It participates in the regulation of biological, abiotic stress responses and developmental physiological processes by regulating the expression of genes. It has been reported that bZIP transcription factor Atf1 is related to the growth, development and pathogenicity of many pathogenic fungi. Transcription factors can specifically bind to cis-acting elements with the participation of other interacting proteins, thereby regulating the expression of target genes. Thus, screening the interacting proteins is of great significance to further understand the regulatory mechanism of bZIP transcription factor. In this study, a bZIP transcription factor CsAtf1 from Colletotrichum siamense(the causative species of rubber tree anthracnose in China)was cloned and the proteins interacting with CsAtf1 were screened and identified. The homologous sequence of Atf1 was searched from the transcriptome database of C. siamense HN08 by local BLAST, using genomic DNA and cDNA as the template for PCR amplification respectively. Sequence analysis revealed that CsAtf1 was 1758 bp, a cDNA of 1611 bp, encoding 535 amino acids, including two introns, three Aft1 domains and one BRLZ domain. Then, the CsAtf1 gene was cloned into the yeast expression vector pGBKT7, and identified by PCR and sequencing. The recombinant bait plasmid pGBKT7-CsAtf1 was transformed into yeast strain Y2 H Gold competent cells. Besides, self-activation and toxicity detection of bait plasmid yeast showed that pGBKT7-CsAtf1 didn’t have self-activation and toxicity in yeast strains. These results demonstrated that the yeast bait protein expression vector was successfully constructed, then yeast two-hybrid(Y2 H) system was adopted to screen interacting proteins of baiting vector pGBKT7-CsAtf1 in cDNA library of C. siamense. The results confirmed that 12 proteins may interact with CsAtf1, including cell wall protein PhiA, rodlet protein, alcohol dehydrogenase, mitochondrial hypoxia res

关 键 词：橡胶树 暹罗炭疽菌 转录因子 互作蛋白 

分 类 号：S763.7[农业科学—森林保护学]