红麻6-磷酸葡萄糖酸脱氢酶（6PDGH）基因克隆及RNAi载体构建

【目的】克隆红麻不育系和保持系6-磷酸葡萄糖酸脱氢酶（6PDGH）基因,并构建其RNAi载体,为进一步研究其在红麻花药发育中的功能奠定基础。【方法】根据红麻花药转录组高通量测序数据结果和生物信息学方法获得6PDGH基因全长c DNA拼接序列,分别以红麻不育系（P3A）和保持系（P3B）的花药反转录c DNA及总DNA为模板克隆红麻6PGDH基因全长。依据RNAi载体设计原则,选取红麻6PGDH基因c DNA序列中段389 bp的特异片段做为RNA干扰片段,构建红麻6PGDH基因的RNAi载体。【结果】克隆获得的红麻不育系（P3A）和保持系（P3B）花药6PGDH基因全长均为1751 bp,均包...

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Published in南方农业学报 Vol. 45; no. 12; pp. 2110 - 2116
Main Author 黄志鹏 赵艳红 明庭会 余明丽 冉珊敏 钱景华 周瑞阳 陈鹏
Format Journal Article
LanguageChinese
Published 2014
Subjects
6-磷酸葡萄糖酸脱氢酶
基因克隆
红麻花药
载体构建
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Summary:【目的】克隆红麻不育系和保持系6-磷酸葡萄糖酸脱氢酶（6PDGH）基因,并构建其RNAi载体,为进一步研究其在红麻花药发育中的功能奠定基础。【方法】根据红麻花药转录组高通量测序数据结果和生物信息学方法获得6PDGH基因全长c DNA拼接序列,分别以红麻不育系（P3A）和保持系（P3B）的花药反转录c DNA及总DNA为模板克隆红麻6PGDH基因全长。依据RNAi载体设计原则,选取红麻6PGDH基因c DNA序列中段389 bp的特异片段做为RNA干扰片段,构建红麻6PGDH基因的RNAi载体。【结果】克隆获得的红麻不育系（P3A）和保持系（P3B）花药6PGDH基因全长均为1751 bp,均包含1458 bp的开放阅读框,编码485个氨基酸残基,无内含子;其碱基序列在不育系和保持系中仅存在两个碱基差异。该基因氨基酸序列（Gen Bank登录号KF964027）与拟南芥、玉米、大豆、苜蓿、三角叶杨、蓖麻的6PDGH同源性分别为75.07%、84.57%、86.50%、87.24%、91.19%和92.01%。成功构建了红麻6PGDH基因的RNAi载体,获得了干扰表达载体p ART27-p KANNIBAL-R1＋2。【结论】成功克隆获得红麻6PGDH基因全长序列,构建的干扰表达载体可用于红麻6PGDH基因的功能研究。
Bibliography:HUANG Zhi-peng, ZHAO Yan-hong, MING Ting-hui, YU Ming-li, RAN Shan-min, QIAN Jing-hua, ZHOU Rui-yang, CHEN Peng （1provincial Key Laboratory of Plant Genetics ＆ Breeding, Guangxi University, Nanning 530005, China; 2Cash Crops Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China）
kenaf（Hibiscus cannabinus L.）; 6PGDH; gene cloning; RNA interference vector construction
Objective】The 6-phosphogluconate dehydrogenase（6PDGH） gene of kenaf sterile line and maintainer line were cloned in present study and its RNAi vector was constructed in order to lay the foundation for further research of this gene function in anther development of kenaf（Hibiscus cannabinus L.）. 【Method】The assembling c DNA sequences of 6PDGH gene were obtained based on high-throughput sequencing data of kenaf anther transcriptome and bioinformatics method. The c DNA and total DNA of CMS P3 A and maintainer line P3 B anthers were used as template to clone the full-length of 6PGDH gene. According to RNAi vector designing
ISSN:2095-1191