烟草类胡萝卜素异构酶基因的克隆及功能研究

Cloning and functional analysis of CRTISO gene in Nicotiana tabacum

为进一步研究烟草类胡萝卜素异构酶(Carotenoid isomerase,CRTISO)基因的功能,从普通烟草(Nicotiana tabacum)的c DNA中成功克隆出CRTISO基因编码区(CDS)全长,长度为1716 bp,Blast结果显示与马铃薯(Solanum tuberosum)和番茄(Solanum lycopersicum)的前番茄红素异构酶(Prolycopene isomerase)基因的相似度达93%。将CRTISO基因的部分序列插入烟草脆裂病毒(Tobacco rattle virus,TRV)载体中,构建重组载体p TRV2-CRTISO。通过TRV病毒诱导的基因沉默(Virus induced gene silencing,VIGS)系统抑制本氏烟草(Nicotiana benthamiana)CRTISO基因的表达,同时利用实时荧光定量PCR(Real-time PCR)检测CRTISO下游基因的表达量变化。结果表明,与对照组相比,p TRV-CRTISO载体侵染的烟草叶片出现黄色斑点;CRTISO基因的沉默导致下游番茄红素ε环化酶(ε-LCY)、番茄红素β环化酶(β-LCY)和胡萝卜素β-环羟化酶(β-OHase)基因表达量降低。对烟草CRTISO基因功能的研究有助于揭示烟草中类胡萝卜素合成代谢途径的调节机制,为选育优质烟草品种奠定理论基础。

CDS of carotenoid isomerase （CRTISO 1716 bp） was obtained from cDNA of Nicotiana tabacum by PCR to explore function of CRTISO. Blast results showed that this gene shared 93% identities with Solanum tuberosum and Solanum lycopersicum. Key nucleotide sequence of CRTISO was inserted into tobacco rattle virus （TRV） vector and recombinant pTRV2-CRTISO vector was constructed. Expression level of CRTISO was suppressed through VIGS system. Plants infiltrated with pTRV-CRTISO grew abnormal leaves which had yellow patches. Results of Real-time PCR showed that the expression level of ε -LCY, β-LCY and β -OHase was depressed due to silence of CRTISO. Findings of this study may contribute to further research in carotenoid biosynthesis and help to lay theoretical foundation for tobacco breeding.