利用病毒诱导的基因沉默(VIGS)技术快速鉴定两个马铃薯晚疫病抗性相关EST片段EL732276和EL732318的功能

马铃薯晚疫病是由致病疫霉Phytophthora infestans(Mont.)deBary]引起的第一大作物病害,目前已严重限制了全球马铃薯的生产和发展。马铃薯晚疫病抗性分子机理的研究一直是抗病育种中的热点问题。本研究利用病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术,在本氏烟草(Nicotina benthamiana)中沉默两个马铃薯晚疫病水平抗性相关的基因片段EL732276和EL732318。目的基因沉默后的烟草接种晚疫病菌P.infestans,根据病斑生长速率LGR(length grow rate)来了解这两个基因是否参与烟草对晚疫病的抗性反应。结果表明,目的基因片段EL732276和EL732318被成功地插入烟草脆裂病毒(Tobacco rattle virus,TRV)载体中。利用携带八氢番茄红素脱氢酶基因(phytoene desaturae,PDS)的TRV重组载体病毒接种烟草,感染病毒的烟草植株表现光漂白现象,说明VIGS体系有效。用携带目的片段的重组载体病毒TRV:EL732276和TRV:EL732318感染烟草植株,一个月后,烟草叶片接种晚疫病菌P.infestans,从接种P.infestans的烟草叶片上可以看出,感染TRV空载体的对照烟草叶片上病斑扩展速率非常缓慢,而目的片段EL732276和EL732318沉默后的烟草叶片可见明显的水浸状病斑和少量白色的晚疫病菌菌丝。进一步的分析表明TRV:EL732276重组载体病毒侵染的烟草接种P.infestans后LGR值极显著上升,TRV:EL732318病毒侵染的烟草接种P.infestans后LGR值显著上升,表明EL732276和EL732318同源基因在烟草中沉默后,烟草对P.infestans的抗性显著降低。研究结果认为,病毒诱导的基因沉默技术可在本氏烟草中初步快速鉴定马铃薯抗病相关基因的功能。

Function of Two Potato ESTs EL732276 and EL732318 Related to Late Blight Resistance Using Virus-induced Gene Silencing(VIGS)

Potato late blight,ranks as world agriculture’s most destructive disease,which are caused by Phytophthora infestans(Mont.) de Bary,and potato production has been seriously limited by this disease.Research on molecular mechanism of resistance against P.infestans is a very important issue in potato breeding program.To date,many ESTs involved in potato late blight resistance have been isolated by the molecular biology methods such as suppression subtractive hybridization,DNA microarray and cDNA-AFLP.To screen and analysis of key candidate genes of potato against P.infestans,functional studies of these ESTs are the most urgent thing.In the present study,two potato ESTs EL732276 and EL732318 which involved in late blight resistance were silenced in Nicotiana benthamiana using virus-induced gene silencing(VIGS).The gene silenced N.benthamiana leaves were inoculated with P.infestans and length grow rate(LGR) of lesions were measured.The RT-PCR results showed that the fragments of candidate genes EL732276 and EL732318 have been successfully cloned into Tobacco rattle virus(TRV) vector.The N.benthamiana leaves infected with TRV carrying the phytoene desaturase gene(PDS) were bleached,indicating that the VIGS system was successful.One month late,the N.benthamiana leaves infected with TRV carrying the genes EL732276 and EL732318 fragment individually were inoculated with P.infestans.The results showed that,compared to the control,LGR values were significantly increased in the EL7322276 and EL732318 silenced N.benthamiana plants,suggesting that P.infestans resistance was dramatically decreased after these two genes silenced in N.benthamiana.This founding was consistent with the results that water soaked lesions and white mycelium obviously observed on EL732276 or EL732318 silenced N.benthamiana leaves after P.infestans inoculation,whereas the lesions developed very slowly on the control leaves which infected with TRV empty vector.The results suggest that functions of potato genes involved in late blight resistance can be rapidly confirmed in N.benthamiana by VIGS technique.