罗汉果生长素响应因子SgARF8基因的克隆及表达分析

Cloning and Expression Analysis of Auxin Response Factor 8 (SgA RF8) Gene in Siraitia grosvenorii

基于转录组Unigene序列信息,结合RACE技术,克隆罗汉果生长素响应因子基因Sg ARF8的全长。采用同源克隆法获取罗汉果Sg ACTIN基因,以该基因为内参,通过实时荧光定量PCR检测Sg ARF8在罗汉果不同器官组织中的时空表达。结果表明:获得Sg ARF8基因c DNA全长序列3 266 bp,Gen Bank登录号KU949383,最长ORF为2 547 bp,编码848 aa的蛋白质,预期分子量94.42 k D,等电点5.67,该编码蛋白具有DBD、CTD和MR等转录因子ARF特征性结构域,其5'端和3'端区域比较保守;克隆得到罗汉果内参基因Actin基因部分序列,命名为Sg ACTIN,Gen Bank登录号为KU949382;实时荧光定量分析表明,罗汉果Sg ARF8在茎、叶、芽、雌蕊、雄蕊、幼果中普遍表达,并且在雌蕊和15 d幼果中表达量较高。可见Sg ARF8广泛参与罗汉果生长发育调节过程,尤其与雌蕊和幼果的器官形态建成密切相关,也暗示该基因在罗汉果性别调控和单性结实遗传改良中具有重要应用价值。

According to the Unigene sequence information from transcriptome data and combined with RACE techniques, the full-length of auxin response factor gene （SgA RF8） ofSiraitia grosvenorii was obtained. Secondly, the Sg, A CTIN gene ofS. grosvenorii was obtained by homologous cloning, which was set as an internal reference gene. Then, the spatio-temporal expression of SgA RF8 in different tissues of S. grosvenorii was analyzed by quantitative real-time PCR. The result showed that the full-length of SgARF8 cDNA was cloned with 3 266 bp （GenBank accession： KU949383）, whose longest open reading frame （ORF） was 2 547 bp, encoding 848 aa protein with MW （Molecular Weight） of 94.42 kD and pI （Isoelectric point） of 5.67. The protein had several characteristic domains of ARF transcript factor, including DBD, CTD, MR conserved domains, and conservative 5＇ and 3＇ end regions. The partial coding sequence of internal reference A ctin gene ofS. grosvenorii was named as Sg, A CTIN （GenBank accession： KU949382）. qRT-PCR analysis indicated that SgARF8 gene was generally expressed in stem, leaf, bud, pistil, stamen and young fruit of S. grosvenorii, with the highest expression in the pistil and young fruits of 15 days. This indicated that the SgARF8 gene was widely involved in the growth and development regulation of S. grosvenorii, and especially it was closely related with the formation of pistil and young fruit, suggesting that it had important value in sex regulation and genetic improvement ofparthenocarpy ofS. grosvenorii.