棉花P-type ATPases基因的克隆及表达分析  

Molecular cloning and expression analysis of P-type ATPases gene from Gossypium barbadense

作　　者：陈菲[1,2] 杨郁文[2] 何冰[2] 陈天子[2] 袁洪波[1,2] 连梓伊[1,2] 张保龙[2] 刘蔼民[2] 

机构地区：[1]南京农业大学农学院,江苏南京210095 [2]江苏省农业科学院,江苏省农业生物学重点实验室,江苏南京210014

出　　处：《江苏农业学报》2011年第6期1192-1197,共6页Jiangsu Journal of Agricultural Sciences

基　　金：转基因专项资助项目(2009ZX08005-003B);江苏省农业科技自主创新基金项目[CX(10)434]

摘　　要：为了探明棉花P-type ATPases基因在植物不同组织和多种逆境条件下的表达情况,利用棉花黄萎病菌的P-type ATPases蛋白序列,在GenBank中搜索棉花EST序列,得到2个同源的棉花EST片段,结合RACE和Genome walking获得了这个基因的完整读码框序列,其开放阅读框长度为3 570 bp,编码1 190个氨基酸,与毛果杨、蓖麻的同源性达86%左右,将其命名为Gbpatp。洋葱表皮亚细胞定位观察结果显示,Gbpatp编码的蛋白分布在细胞膜上。RT-PCR技术检测组织表达特异性分析表明,P-type ATPases在茎和棉絮中的表达量较高,在种子中低水平表达,在叶片和花蕾中表达量极低,说明Gbpatp可能与棉花较成熟的器官茎、棉絮和种子的生长发育密切相关。在逆境胁迫中发现,干旱处理后Gbpatp的表达强烈,并且随处理时间延长表达量逐渐增加;重金属Cu2+处理24 h后Gbpatp表达量升高,但48 h急剧下降;Gbpatp在低温处理下也有轻微表达。激素诱导后发现,Gbpatp对赤霉素和脱落酸均有响应,随胁迫时间延长其表达量仍能维持在一定水平。To investigate the expression of P-type ATPases in different tissues under abiotic stress conditions,two ESTs homologic to P-type ATPases gene from Verticillium dahliae Kleb.were found through Blast in GenBank database.The gene was named Gbpatp,and its full open reading frame was obtained by RACE and Genome walking,being 3 570 bp of the length,and encoding a polypeptide of 1 190 amino acids.It shared about 86% homology to its homologous nucleotide sequence of Populus trichocarpa or Ricinus communis.Subcellular localization result showed that the protein encoded by Gbpatp distributed in cell membrane.Its distribution in different organ was analyzed by semi-quantitative RT-PCR.Gbpatp was abundant in stem and fiber,low in seed,and scarce in leaf and flower bud,which suggested that Gbpatp might be insubstantial connection with comparative mature organs.The expressions of Gbpatp remarkably increased under drought stress with the extension of time.It increased 24 h after CuSO4 stress,and decreased after 48-h CuSO4 stress.It showed a weak expression under low temperature stress.The expression of Gbpatp also responded to gibberellic acid or abscisic acid induction,and kept in a certain level with the extension of time.

关 键 词：棉花 P-TYPE ATPases基因 基因表达 克隆 

分 类 号：Q78[生物学—分子生物学]