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作物学报 ›› 2011, Vol. 37 ›› Issue (02): 362-368.doi: 10.3724/SP.J.1006.2011.00362

• 研究简报 • 上一篇    下一篇

陆地棉两个同源基因GhBlind的克隆与表达分析

熊冠军1,徐芹1,华金平1, 2, *   

  1. 1 中国农业大学农学与生物技术学院，北京100193；2中国农业大学 / 作物基因组学与遗传改良农业部重点开放实验室 /杂种优势研究与利用教育部重点实验室 / 作物遗传改良北京市重点实验室，北京100193
  • 收稿日期:2010-06-23 修回日期:2010-09-27 出版日期:2011-02-12 网络出版日期:2010-11-16
  • 通讯作者: 华金平, E-mail: jinping_hua@cau.edu.cn
  • 基金资助:

    本研究由教育部新世纪优秀人才支持计划项目(NCET-06-0106)，国家高技术研究发展计划(863计划)重大专项(2009AA101104)，教育部长江学者和创新团队发展计划和高等学校学科创新引智计划(111-2-03)资助。

Isolation and Expression Analysis of Two GhBlind Homologs in Upland Cotton (Gossypium hirsutum L.)

XIONG Guan-Jun1,XU Qin1,HUA Jin-Ping1,2,*   

  1. 1 College of Agronomy & Biotechnology, China Agricultural University, Beijing 100193, China；2 Key Laboratory of Crop Genetic Improvement and Genome of Ministry of Agriculture / Key Laboratory of Crop Heterosis and Utilization of Ministry of Education / Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing 100193, China
  • Received:2010-06-23 Revised:2010-09-27 Published:2011-02-12 Published online:2010-11-16
  • Contact: HUS Jin-Ping, E-mail: jinping_hua@cau.edu.cn

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摘要： Blind同源基因对植物株型调控起着重要作用。本研究用同源克隆策略，在陆地棉(Gossypium hirsutum L.) 腋芽部位cDNA中克隆出2个Blind同源基因GhBlind1(GenBank登录号为HQ115643)和GhBlind2(登录号为HQ115644)。GhBlind1和GhBlind2由3个外显子和2个内含子组成，分别编码359个和262个氨基酸残基。组织特异性表达分析表明, GhBlind1在腋芽部位和茎尖分生组织部位优势表达，在根、叶、茎尖、幼嫩纤维表达，而在茎部不表达；GhBlind2在腋芽部位以及根、茎、叶片、茎尖表达，而在纤维不表达。通过重组PCR技术，构建表达载体Psuper-gus-b1和Psuper-gus-b2；应用根癌农杆菌EHA105介导转化棉花胚性愈伤组织进行瞬时表达分析。将棉花胚性愈伤组织共培养4 d后，2个表达载体转化的胚性愈伤组织中均能检测到GUS活性，但Psuper-gus-b2表达活性强于Psuper-gus-b1。对Blind同源蛋白的保守结构域比对发现，GhBlind1和GhBlind2与Blind同源蛋白的一致性分别达94%和91%。以Blind同源蛋白作为外参，结合NCBI已公开的23个棉花MYB蛋白构建系统发生树，发现GhBlind1和GhBlind2与棉花大多数MYB蛋白遗传距离较远，和Blind同源蛋白组成一个较小的分支。

关键词: 棉花, Blind, 同源克隆, 表达分析

Abstract: In higher plants, Blind homologs play an important role in plant architectures. Two Blind orthologs, GhBlind1 and GhBlind2, were isolated from cDNA of the shoot apex of upland cotton (Gossypium hirsutum L.) by homologous cloning strategy. GenBank accession number is HQ115643 for GhBlind1, and HQ115644 for GhBlind2. Similar to other Blind homologs, GhBlind1 and GhBlind2 were consisted of three exons and two introns, and encoded 359 and 262 amino acids, respectively. Based on the RT-PCR analysis, it was found that GhBlind1 expressed in root, leaf, and tender fiber, and predominantly in shoot apical meristem (SAM) and shoot apex, but not in stem; while GhBlind2 expressed in root, stem, leaf, SAM and shoot apex, but not in tender fiber. Using recombinant PCR technology, expression vectors Psuper-gus-b1 and Psuper-gus-b2 were constructed and used for Agrobacterium-mediated transient expression by transformation of embryogenic callus of upland cotton cv. Coker 201. Histochemical assays of GUS activity showed that after co-cultivation of embryogenic callus for four days, positive events were more obvious in Psuper-gus-b2 than in Psuper-gus-b1. The alignments between GhBlind1, GhBlind2 and the domain of Blind homologs showed the identities of 94% and 91%, respectively. The results of phylogenetic tree showed that GhBlind1 and GhBlind2 were genetically divergent from most of the other R2R3-MYB members in Gossypium hirsutum L. We speculated that GhBlind1 and GhBlind2 are probably related with the development of lateral branch in upland cotton.

Key words: Upland cotton, Blind, Homologous isolation, Expression profile

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