Ding Zhong-Tao, Zhang Zhi, Luo Di, Zhou Jin-Yan, Zhong Juan, Yang Jie, Xiao Liang, Shu Dan, Tan Hong
Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, the Chinese Academy of Sciences, No. 9 Section 4, Renmin Nan Road, Chengdu 610041, China.
University of the Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China.
Int J Mol Sci. 2015 May 6;16(5):10301-23. doi: 10.3390/ijms160510301.
The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain.
植物病原子囊菌灰葡萄孢产生几种具有生物技术意义的次生代谢产物，尤其已被用于工业规模生产S-(+)-脱落酸。为了利用根癌农杆菌介导的转化系统调控灰葡萄孢特定次生代谢产物生物合成基因的表达水平，采用In-Fusion组装方法构建了两个表达载体（具有不同选择标记的pCBh1和pCBg1）和一个RNA沉默载体pCBSilent1。两个表达载体在组成型表达增强绿色荧光蛋白（eGFP）方面都非常有效，并且pCBSilent1有效地沉默了灰葡萄孢中的eGFP基因。然后，利用这些反向遗传学工具，将灰葡萄孢中一个被认为参与脱落酸生物合成的基因Bcaba4作为基因过表达和RNA沉默的靶点。bcaba4的过表达显著诱导了灰葡萄孢野生型菌株Bc-6中脱落酸的形成，而bcaba4的基因沉默显著降低了一个能产生脱落酸的灰葡萄孢菌株中脱落酸的产量。
