HSP70基因参与粉红螺旋聚孢霉寄生核盘菌机制研究

Clonostachys rosea (syn. Gliocladium roseum) is a mycoparasite which has showed great potential for biocontrol of various fungal plant diseases. In previous study, we sequenced the transcriptome of C. rosea 67-1 parasitizing sclerotia of Sclerotinia sclerotiorum and obtained a heat shock protein 70 (HSP70) encoding gene crhsp70, which significantly upregulated expressed under sclerotia induction by using qRT-PCR. HSP70 is a structurally and evolutionarily conserved chaperone protein that participates in protein folding, cooperative immune, cell differentiation and death regulation. Resent research shows that HSP70 is involved in the recognition signaling to host. However, the mechanism of HSP70 in parasitism of mycoparasites is unknown yet. In this study, full-length sequence of crhsp70 will be cloned and the bioinformation will be analyzed. Gene knockout and complementation, as well as the biocontrol efficacy of the mutants in greenhouse will be conducted to clarify the function of crhsp70 during C. rosea parasitizing S. sclerotiorum. The transcriptome of the wild type and crhsp70-deficient mutant will be sequenced and differentially expressed genes will be analyzed to investigate the mechanism in which crhsp70 involves during mycoparasitic process of C. rosea. This research is of great significance for investigating the biocontrol mechanism of C. rosea.

粉红螺旋聚孢霉是多种重要植物病原真菌的菌寄生菌，具有较强的生防应用潜力。项目组通过对其高效菌株67-1寄生核盘菌菌核转录组测序分析和实时荧光定量监测，筛选获得了一个在菌核诱导条件下显著上调表达的热激蛋白70（HSP70）编码基因crhsp70。HSP70是一类在结构和进化上都非常保守的分子伴侣蛋白，可以参与到蛋白质折叠、协同免疫、调控细胞分化和凋亡等过程。最新研究表明，HSP70还与血吸虫对宿主的识别信号相关，但其在菌寄生菌中的作用机制迄今尚不明确。本项目拟根据67-1全基因组信息对crhsp70进行全长克隆和生物信息学分析；通过基因敲除、回补和温室防病效果验证crhsp70在粉红螺旋聚孢霉寄生核盘菌过程中的作用；进而通过对野生菌株和基因敲除突变株寄生核盘菌菌核转录组测序，分析差异基因，阐明crhsp70参与粉红螺旋聚孢霉寄生核盘菌机制；对揭示粉红螺旋聚孢霉生防作用机制具有重要意义。

粉红螺旋聚孢霉是一类非常重要的生防菌寄生菌，能够侵染丝核菌和核盘菌等多种植物病原真菌，具有优良的生防能力。热激蛋白70是一类在结构和进化上都非常保守的分子伴侣蛋白，可以参与到蛋白质折叠、协同免疫、调控细胞分化和凋亡等过程，但是其在菌寄生过程中所起到的作用未知。本项目以粉红螺旋聚孢霉全基因组中编码热激蛋白70的基因crhsp70为出发点，对crhsp70进行克隆和生物信息学分析，并利用分子生物学手段研究了其在粉红螺旋聚孢霉寄生核盘菌过程中所起的作用。研究表明crhsp70会显著影响粉红螺旋聚孢霉对核盘菌的寄生能力和对大豆菌核病的防治能力。为进一步了解crhsp70参与粉红螺旋聚孢霉寄生核盘菌的机制，本项目对粉红螺旋聚孢霉野生菌株和crhsp70敲除突变株寄生核盘菌菌核的转录组进行了测序和分析。结果表明crhsp70敲除后大量基因显著下调表达，其中编码细胞色素P450和细胞壁降解酶的基因与产生毒素和细胞壁降解酶相关。我们推测crhsp70通过影响细胞色素P450和细胞壁降解酶编码基因的表达从而影响了粉红螺旋聚孢霉对核盘菌的寄生能力。本研究为进一步明确粉红螺旋聚孢霉生防机制提供了基础。