利用VIGS技术在本氏烟中沉默枸杞PDS同源基因的研究

Silencing Lycium PDS Ortholog in Nicotiana benthamiana Using VIGS

【目的】本项研究旨在通过建立一个异源VIGS体系,为枸杞重要性状相关基因的功能鉴定提供前期信息。【方法】从枸杞叶片中克隆获得409 bp长的八氢番茄红素脱饱和酶基因(phytoene desaturase,PDS)的编码区片段,构建入基于烟草脆裂病毒的VIGS空载体中,通过农杆菌介导的侵润接种,将重组的病毒沉默载体pTRV2-PDS转入本氏烟中。【结果】本氏烟幼苗被侵染接种后,沉默处理植株的叶片呈现整叶白化的表型,而阴性对照植株叶片一直未出现变白的症状,qRT-PCR检测表明,接种17 d后,与阴性对照植株相比,沉默处理植株PDS基因的表达显著地被抑制(P<0.05),说明枸杞PDS基因的同源基因在本氏烟中得到了有效沉默。【结论】该VIGS体系的构建,可用于对枸杞部分基因功能的快速初步鉴定和筛选,旨在提高枸杞基因功能研究的效率。

[Object]This study aims to provide preliminary information for the functional identification of genes related to important traits of Lycium by establishing a heterologous VIGS system.[Method]The main methods include a 409 bp phytoene desaturase(PDS)coding region fragment cloned from the leaves of Lycium,which was inserted into the tobacco rattle virus-based VIGS empty vector.The recombinant virus silencing vector pTRV2-PDS was transferred into Nicotiana benthamiana by agrobacterium infiltration.[Result]After the Nicotiana benthamiana seedlings were inoculated,the leaves of the silenced plants showed the phenotype of whole-leaf photobleaching,while the leaves of the negative control plants did not show any signs of photobleaching.The qRT-PCR of the target gene showed that the expression of PDS gene in silenced plants was significantly suppressed(P<0.05)at 17 days post infiltration compared with the negative control plants.The study showed that the ortholog of Lycium PDS gene was effectively silenced in Nicotiana benthamiana.[Conclusion]The heterologous VIGS system can be used for the rapid preliminary functional identification and screening of some gene of Lycium,which is helpful to improve the efficiency of research on gene function of Lycium.