【摘要】 病毒诱导的基因沉默(VIGS)是研究植物基因功能的一种非常有用的技术,它可随意敲掉感兴趣的基因,耗时较短,不需建立稳定的遗传体系.本研究利用VIGS方法,以紫花地丁的VpTM6-2作为探针序列,去沉默本氏烟草中的内源NbTM6,以探讨VpTM6-2的生物功能.VpTM6-2属于B类MADS-box基因,文中设计的探针序列长357bp,与NbTM6相应的序列之间具有69.0%的同源性,除一个碱基有差异外,存在一段连续长27个碱基的100%同源序列.结果表明,VpTM6-2能够特异性敲除本氏烟草的内源NbTM6,使得NbTM6的表达明显下降,花粉发育异常(其余花器官发育均正常).本氏烟草VIGS体系使得紫花地丁中的一些基因可在异源体系中得到功能验证.该研究进一步证明VpTM6-2表达含量的下降可能与紫花地丁两型花发育有一定的关系.更多还原

【Abstract】 Virus-induced gene silencing(VIGS) is a particularly useful tool for plant functional genomics because it can knock down the genes of interest,the observation of elicited phenotypes needs a short time,and does not require the genetic transformation of plants.In order to investigate the function of VpTM6-2,the gene silencing efficiency of the corresponding orthologs NbTM6 is determined by using the coding region of VpTM6-2 as triggers in N.benthamiana through VIGS.VpTM6-2 belongs to the B-class MADS-box genes.The VpTM6-2 trigger sequence is 357 bp long and has 69.0% identity at the nucleotide level with the putative target sequences of NbTM6,and there is a 27-nucleotide stretch in VpTM6-2 and NbTM6 showing 100% identity with one base mismatch.The result showed that the NbTM6 is effectively knocked down by the VpTM6-2 trigger sequence in N.benthamiana.The pollen viability in N.benthamiana flowers is reduced,and the expression of NbTM6,which has the highest identity with VpTM6-2,is significantly reduced in the abnormal flowers of N.benthamiana compared to the wild-type flowers.This work provides a system that heterologous sequence-triggered VIGS is an efficient way to investigate functional conservation of orthologous genes.The study further showed that the decline of VpTM6-2 expression in floral tissue of V.philippica is related with the CH-CL flower development.更多还原