矮牵牛PDS基因的克隆及其在shRNA介导的基因沉默中的应用

发布时间：2018-01-31 14:58

本文关键词： 矮牵牛八氢番茄红素脱氢酶基因基因沉默 shRNA　出处：《园艺学报》2017年02期 　论文类型：期刊论文

【摘要】：以矮牵牛(Petunia hybrida)自交系‘Mitchell Diploid’(MD)为材料,通过3′RACE和5′RACE获得了其八氢番茄红素脱氢酶基因(PhPDS)的cDNA序列,进而通过PCR扩增出编码区的基因组序列。分析结果显示,PhPDS的cDNA序列全长2 182 bp,编码区序列1 746 bp,编码582个氨基酸。推测的蛋白质序列具有类胡萝卜素脱氢酶的保守结构域特征。编码区的基因组序列长7 609 bp,结构与番茄和拟南芥等PDS基因相似,具有14个外显子和13个内含子。利用博德研究所的小发夹RNA(shRNA)设计程序设计了两个针对PhPDS的shRNA,构建植物表达载体转化矮牵牛后,从其中一个载体的转化子中获得白色愈伤组织和幼枝,对白色幼枝的RT-PCR检测表明,其PDS基因的cDNA累积量减少,表明shRNA基因沉默技术能在矮牵牛中应用。以PDS基因为靶基因比用花色素合成基因研究基因沉默技术能更快地分析基因沉默的效果,全长序列的获得将有助于PDS基因在矮牵牛基因沉默技术研究中的应用。
[Abstract]:Petunia hybrida, an inbred line, was used as material. The cDNA sequence of its octahydrolycopene dehydrogenase gene, PhPDS, was obtained by 3H race and 5H race. Then the genomic sequence of the coding region was amplified by PCR. The results showed that the cDNA sequence of PhPDS was 2 182 BP in length and the coding region was 1 746 BP. It encodes 582 amino acids. The deduced protein sequence has the conserved domain of carotenoid dehydrogenase. The genome sequence of the coding region is 7 609 BP long. The structure is similar to the PDS gene of tomato and Arabidopsis thaliana. With 14 exons and 13 introns, two shRNA targeting PhPDS were designed using the Bod Institute's small hairpin RNA-shRNAs. After the plant expression vector was constructed and transformed into petunia, the white callus and young branches were obtained from the transformants of one of the vectors, and the RT-PCR detection of white young shoots showed that. The cDNA accumulation of PDS gene decreased. The results showed that shRNA gene silencing technique could be used in Petunia hybrida, and the effect of gene silencing could be analyzed more quickly by using PDS gene as target gene than by using anthocyanin synthesis gene to study gene silencing. The acquisition of full-length sequence will be helpful to the application of PDS gene in the study of petunia gene silencing.
【作者单位】：西南大学园艺园林学院重庆市花卉工程技术中心南方山地园艺学教育部重点实验室;
【基金】：中央高校基本科研业务费专项资金项目(XDJK2016D062) 重庆市科委项目(cstc2014yykf B80008)
【分类号】：S681.9
【正文快照】： 类胡萝卜素是光合作用过程中的辅助色素,能够猝灭单线态氧从而保护叶绿素不受到伤害(Qin et al.,2007)。八氢番茄红素脱氢酶(phytoene desaturase,PDS)是类胡萝卜素合成途径中的关键酶,PDS基因功能的失活会导致类胡萝卜素不能有效合成,叶绿素在光照条件下会被破坏,使本应呈绿

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