矮牵牛PDS基因的克隆及其在shRNA介导的基因沉默中的应用

Cloning of Petunia PDS Gene and Its Application in shRNA-mediated Gene Silencing

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。

The full-length cDNA sequence of the phytoene desaturase gene（PDS）of Petunia hybrida was isolated by using 3′ RACE and 5′ RACE. Subsequently,the genomic sequence of the PhPDS coding region was obtained by PCR amplification using primers designed according to its cDNA sequence. Sequence analysis indicated that the full-length cDNA of PhPDS was 2 182 bp in length,containing a coding sequence（CDS）of 1 746 bp coding for a putative protein of 582 amino acids. The putative PhPDS protein is characterized by the two conserved domains of the carotenoids dehydrogenase family. The genomic sequence of PhPDS coding region was 7 609 bp,including 14 exons and 13 introns. This structure is similar to that of tomato and Arabidopsis PDS genes. Two shRNAs targeted to Ph PDS were designed using the TRC shRNA design process from Broad Institute,and plant expression vectors（pGSH-pds1 and pGSH-pds2）were constructed using conventional molecular cloning technique. Albino callus and shoots were produced from pGSH-pds1 transformants after Agrobacterium tumefaciensmediated transformation of petunia leaf discs. RT-PCR analysis showed that the accumulation of PDS m RNA in albino shoots was reduced clearly,which indicating that shRNA technique is applicable to petunia. CHS（chalcone synthase）is conventionally employed to evaluate gene silencing methods in petunia. However,phenotype caused by the disruption of PDS function can be discerned earlier than that of CHS. Cloning of petunia PDS will facilitate its application in gene silencing research.