忍冬VIGS基因沉默体系构建

目的 以忍冬Lonicera japonica八氢番茄红素脱氢酶（phytoene desaturase，PDS）基因为标记基因，利用ZMBJ-CMV载体建立基于病毒诱导的忍冬基因沉默体系。方法 以忍冬为材料，通过选取同源性较高的片段设计PDS基因的特异性引物，克隆出片段长度为135 bp的序列作为目的基因片段，经双酶切后与黄瓜花叶病毒载体连接构建VIGS重组载体，转化根癌农杆菌后，侵染忍冬幼苗。此外，还对农杆菌吸光度（A）值和侵染方法对PDS基因沉默效率的影响进行了比较。结果 被侵染植株叶片PDS基因表达水平显著降低，叶绿素和类胡萝卜素含量显著下降，叶片出现白化表型。结论 建立了以ZMBJ-CMV载体诱导的忍冬基因沉默体系，农杆菌A值为0.8、注射后再进行真空浸润，能够提高忍冬目标基因的沉默效率，为开展忍冬关键基因的功能研究提供了技术支撑。

Construction of VIGS gene silencing system in Lonicera japonica

Objective The ZMBJ-CMV vector was used to establish the gene silencing system of Lonicera japonica based on virus induction by using the phytoene desaturase (PDS) gene as the marker gene. Methods Using L. japonica as the material, specific primers of PDS gene were designed by selecting fragments with high homologous characteristics, and the sequence length of 135 bp was cloned as the target gene fragment. After double enzyme cutting, this sequence was ligated to cucumber mosaic virus to construct VIGS recombinant vector, which was transformed to Agrobacterium tumefaciens. Finally, the Agrobacterium tumefaciens was used to infect the L. japonica seedlings. At the same time, the effects of Agrobacterium tumefaciens absorbance (A) value and infection method on the silencing efficiency of PDS gene were also compared. Results The expression level of PDS gene in the leaves of infected plants decreased significantly, and the contents of chlorophyll and carotenoid also decreased significantly. Moreover, the leaves of L. japonica showed albino phenotype. Conclusion The gene silencing system of L. japonica induced by ZMBJ-CMV vector was established. The A value of Agrobacterium tumefaciens was 0.8, and vacuum infiltration after injection could improve the silencing efficiency of L japonica target gene. This study provides technical support for the functional study of key genes in L. japonica.