关键词: 矮牵牛, 八氢番茄红素脱氢酶基因, 基因沉默, shRNA

Abstract:

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 PhPDS 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 tumefaciens- mediated transformation of petunia leaf discs. RT-PCR analysis showed that the accumulation of PDS mRNA 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 applicationin gene silencing research.

Key words: petunia, phytoene desaturase gene, gene silencing, shRNA