红麻6-磷酸葡萄糖酸脱氢酶(6PDGH)基因克隆及RNAi载体构建

6-phosphogluconate dehydrogenase (6PDGH) gene cloning of kenaf (Hibiscus cannabinus L.) and its RNA interference vector construction

【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。

【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 rule, the 389 bp gene-specific fragment of 6PGDH c DNA sequence was used as RNA interference fragment to insert into p KANNIBAL vector in forward and re-verse direction, respectively, to construct the RNAi vector. 【 Result 】 The full-length of cloned 6PGDH gene in P3 A and P3 B anthers was 1751 bp in length, which contained a 1458 bp open reading frame（ORF） encoding 485 amino acids and had no intron. Only two bases ＇ differences were found between base sequence of CMS and maintainer line.The amino acid sequence of 6PGDH gene shared 75.07%, 84.57%, 86.50%, 87.24%, 91.19% and 92.01% of homology with Arabidopsis thaliana（AY084486）, Zea mays（AF061838）, Glycine max（B007907）, Medicago sativa（U18239）, Populus trichocarpa（XM_002329211）, Ricinus communis（XM_002530757）. The RNAi vector of 6PGDH gene in kenaf was constructed successfully to obtain the interference expression vector p ART27-p KANNIBAL-R1 ＋2.【Conclusion 】The full-length sequence of 6PGDH gene in kenaf was cloned successfully, and the constructed RNA interference vector p ART27-p KANNIBAL-R1＋2 could be used for studying 6PGDH gene function of kenaf.