陆地棉环核苷酸离子通道蛋白基因GhCNGC8克隆及初步表达分析  

Molecular Cloning and Preliminary Expression Analysis of GhCNGC8 Gene in Gossypium hirsutum

作　　者：赵准 胡文冉[1] 邵武奎 黄全生[1] ZHAO Zhun;HU Wenran;SHAO Wukui;HUANG Quansheng(Institute of Nuclear Technology and Biotechnology,Xinjiang Academy of Agricultural Sciences,Key Laboratory of Crop Biotechnology of Xinjiang Uygur Autonomous Region,Urumqi 830091,China;College of Life Sciences,Xinjiang Agricultural University,Urumqi 830052,China)

机构地区：[1]新疆农业科学院核技术生物技术研究所,新疆维吾尔自治区农作物生物技术重点实验室,乌鲁木齐830091 [2]新疆农业大学生命科学学院,乌鲁木齐830052

出　　处：《西北农业学报》2024年第12期2306-2314,共9页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：新疆重点实验室开放课题项目(2022D04008);省部共建干旱荒漠区作物抗逆遗传改良与种质创新国家重点实验室培育建设项目(ZYYD2022B07)。

摘　　要：为明确环核苷酸门控离子通道蛋白基因GhCNCG8对棉花抗逆方面的作用。利用棉花叶片克隆获得该基因,生物信息学方法分析其理化性质、结构和进化关系,利用qRT-PCR(实时荧光定量)方法分析基因在黄萎病菌、干旱、盐胁迫以及激素诱导下GhCNCG8表达模式和组织表达特异性;构建该基因VIGS沉默载体,利用农杆菌介导方法转化该基因到棉花叶片中,荧光定量PCR方法检测沉默效率。以棉花叶片cDNA为模板成功克隆GhCNGC8基因,该基因开放阅读框为2247 bp,编码748个氨基酸。进化树分析结果表明,陆地棉GhCNGC8基因与拟南芥AtCNGC8基因关系较近,多序列结果比对显示,GhCNGC8蛋白结构符合CNGCs家族蛋白结构特征。qRT-PCR结果表明,该基因对黄萎病、干旱、盐处理等胁迫及茉莉酸(JA)、水杨酸(SA)和双氧水(H_(2)O_(2))处理均有一定程度的响应,qRT-PCR分析结果表明,GhCNGC8基因在根、茎、叶、花、萼片和苞片中均有表达,叶中表达量最高。构建该基因的VIGS沉默载体转化棉花,qRT-PCR结果表明,该基因在棉花中已经沉默,表达量相比对照植株降低70%左右,表明GhCNGC8沉默载体构建成功并在棉花体内正常运行,GhCNGC8可能在棉花抗逆方面发挥重要作用。To investigate the role of the GhCNGC8 gene in cotton stress resistance,we cloned it from cotton leaves using PCR and utilized bioinformatics methods to analyze its physicochemical properties,structure,and evolutionary relationships.We used qRT-PCR to analyze the expression patterns of GhCNGC8 under various various stress conditions,including infection by Fusarium wilt pathogen,drought,salt stress,and hormone induction,as well as its tissue-specific expression.We constructed a VIGS vector targeting this gene and transformed it into cotton leaves using Agrobacterium-mediated methods.The gene silencing efficiency was assessed using quantitative fluorescence PCR.The GhCNGC8 gene was successfully cloned from cotton leaf cDNA,possessing an open reading frame of 2247 bp that encodes 748 amino acids.Phylogenetic analysis indicated a close relationship between the upland cotton GhCNGC8 gene and the Arabidopsis AtCNGC8 gene.Multiple sequence alignment revealed that the GhCNGC8 protein conforms to the structural characteristics of CNGC family proteins.qRT-PCR results showed that the gene responded to Fusarium wilt,drought,salt stress,as well as jasmonic acid,salicylic acid,and hydrogen peroxide treatments to varying degrees.The GhCNGC8 gene expression was detected in roots,stems,leaves,flowers,sepals,and bracts,with the highest expression in leaves.Transformation of the GhCNGC8 gene VIGS silencing vector into cotton resulted in successful silencing,with expression levels reduced by approximately 70%compared to control,confirming the successful construction and normal operation in cotton.GhCNGC8 may play a significant role in cotton stress resistance.

关 键 词：棉花 GhCNGC8 基因克隆 表达分析 载体构建 

分 类 号：S562[农业科学—作物学]