芥菜锌指蛋白转录因子基因Bj26的克隆与鉴定  

Cloning and Characterization of Brassica juncea Zinc Finger Protein Transcription Factor Gene Bj26

作　　者：贾双伟 高英[1] 赵开军[1] 

机构地区：[1]中国农业科学院作物科学研究所,农作物基因资源与基因改良国家重大科学工程,北京100081

出　　处：《作物学报》2014年第7期1174-1181,共8页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(31071484)资助

摘　　要：锌指蛋白是一类重要的转录因子家族,参与植物基因转录调节、发育及胁迫反应等生理过程。我们前期研究发现芥菜诱导型启动子BjC-P的W-box-like-4为其真菌诱导的核心元件,本研究通过酵母单杂交技术从芥菜cDNA文库中筛选到与W-box-like-4序列特异互作的转录因子基因Bj26。生物信息学分析表明,Bj26含735 bp的开放阅读框,编码一个新的C2H2型锌指蛋白,包括2个典型的C2H2型锌指结构及2个植物特有的QALGGH氨基酸保守序列,等电点pI为9.2,分子量为26.6 kD。亚细胞定位显示该蛋白位于细胞核。本氏烟瞬时表达分析表明Bj26蛋白通过与BjC-P的真菌诱导核心元件序列特异互作,激活启动子BjC-P。实时荧光定量PCR结果显示Bj26在真菌诱导下表达量明显增高。Bj26的CDS序列与拟南芥和水稻中的C2H2型锌指蛋白序列比对及进化树分析表明,Bj26与拟南芥的C2H2型蛋白同源性高于水稻。以上结果揭示Bj26蛋白可能介导BjC-P真菌诱导响应并参与植物抗真菌病原菌的调控过程。Zinc-finger proteins, forming an important transcriptional factor family, are involved in gene transcriptional regulation, development and stress-responses in plants. The previous studies revealed that BjC-P is a fungus-inducible promoter from Brassicajuncea and W-box-like-4 is the core element responsive to fungal infection. Here, we report the molecular cloning and characterization of a zinc finger protein-encoding gene （designated Bj26）. Bj26 was screened out from the Brassica juncea cDNA library by the Yeast One-Hybrid System. Bioinformatic analysis showed that Bj26 contains open reading frame of 735 bp that encodes a new C2H2-type zinc finger protein with an isoelectric point （pI） of 9.2 and a molecular weight of 26.6 kD. The protein consists of two typical zinc-finger domains and contains two conserved QALGGH amino acid sequences. Subcellular localization showed that Bj26 is located in the nuclear. The histochemical and quantitative GUS assays, through transient gene expression in Nicotiana benthamiana leaf, showed that Bj26 can bind to the core element W-box-like-4 and activate the function of BjC-P. qRT-PCR analysis showed that the gene Bj26 expression obviously increased under the induction of the fungal elicitor （Hexa-N-Acetyl-Chitohexaose）. CDS alignments and phylogenetic analysis of Bj26 and other C2H2-type proteins from Arabi- dopsis thaliana （At） and Oryza sativa （Os） （MEGA 6） showed Bj26 shares high similarity with that from Arabidopsis thaliana （At）. All of the above results suggested that Bj26 protein mediates the process of plant response to fungal pathogen.

关 键 词：酵母单杂交 C2H2锌指蛋白 Bj26基因 真菌诱导 瞬时表达 实时荧光定量PCR 

分 类 号：S637[农业科学—蔬菜学] Q943.2[农业科学—园艺学]