膜泡运输蛋白VdSec22参与大丽轮枝菌胞外蛋白的分泌和致病性

Vesicular transport protein VdSec22 is involved in secretion of extracellular protein and pathogenicity in Verticillium dahliae

摘要:

摘要:【目的】验证大丽轮枝菌分泌途径中一个膜泡运输蛋白VdSec22的功能，为防治棉花黄萎病提供潜在的生物靶点。【方法】利用“正负双向筛选法”的方法，构建大丽轮枝菌VdSec22蛋白编码基因缺失的突变体菌株ΔQF。通过农杆菌介导的转化，将其编码基因VdSec22重新导入ΔQF构建了功能回补菌株CΔQF。以野生型菌株为对照，检测上述菌株分泌胞外蛋白(果胶酶、纤维素酶和毒素蛋白)的能力;采用蘸根接种的方法，检测上述菌株对棉花致病性的差异。同时通过定量PCR检测内质网分子伴侣表达量的方法，推断突变体菌株ΔQF中是否发生了内质网应激反应。【结果】成功构建了基因敲除突变体ΔQF和功能回补菌株CΔQF。突变体菌株ΔQF的果胶酶、纤维素酶、毒素蛋白的分泌能力和对棉花的致病性均较野生型减弱，并且产生了内质网应激反应。重新导入VdSec22基因可弥补突变体菌株ΔQF的上述缺陷。【结论】VdSec22是大丽轮枝菌的一个重要分泌途径蛋白，在大丽轮枝菌诸多胞外致病蛋白的分泌和棉花致病性中起重要作用。VdSec22可作为防治棉花黄萎病的潜在生物靶点。

关键词:关键词:大丽轮枝菌，分泌蛋白，分泌途径，基因敲除

Abstract:

Abstract:［Objective］The aim of this study was to characterize VdSec22 of Verticillium dahliae，which is an intracellular vesicle fusion protein involved in fungal secretory pathway，and to provide a potential gene target for controlling Verticillium wilt disease.［Methods］VdSec22 deletion mutant ΔQF and functional complementation strain CΔQF by reintroducing the VdSec22 intoΔQF were constructed．Secretion ability of extracellular protein (including pectinase，cellulose，and phytotoxin protin) and pathogenicity of ΔQF and CΔQF were studied compared with that of wild type strain Vd991． Expression level of ER molecular chaperones by quantitative PCＲ was also performed to infer whether EＲ stress was induced in ΔQF．［Results］We successfully constructed VdSec22 deletion mutant strain ΔQF and functional complementation strain CΔQF．VdSec22 deficiencies did disturb secretion ability of extracellular protein such as pectinase，cellulose，and phytotoxin protin．Pathogenicity of ΔQF was dramatically reduce accordingly． We also found loss of VdSec22 resulted in ER stress in V． dahliae cells． Reintroducing functional VdSec22 into ΔQF can compensate for the deficiencies mentioned above．［Conclusion］ VdSec22 is an important secretion pathway protein involved in secretion of extracellular protein and pathogenicity in V．dahliae． VdSec22 provides a potential gene target for controlling the devastating disease．

Key words:Keywords: Verticillium dahliae，secretory protein，secretion pathway，gene knockout