棉花P-typeATPases基因的克隆及表达分析

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3570bp,编码1190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24h后Gbpatp表达量升高,但48h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 VerticUlium dahliae Kleb. Were found through Blast in CenBank database. The gene was named Gbpaip, 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. Subcelhilar 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 in-substantial 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.