【作者】 肖红利；

【导师】 戴小枫；

【作者基本信息】 中国农业科学院 ， 生化与分子生物学， 2014， 博士

【摘要】 本研究将大丽轮枝菌在棉花组织诱导和非诱导（查比克基础培养）体系中分别进行培养，观察其生长表型，提取分泌蛋白并进行致病性研究。结果表明棉花组织诱导培养基更适合大丽轮枝菌的生长，能够促进它产生更多的分生孢子，向培养上清液中分泌更多的蛋白质，胞外蛋白接种棉花能够引起更加典型的黄萎病症。可见，棉花组织诱导体系可以诱导大丽轮枝菌分泌型致病蛋白的表达，通过其分泌蛋白谱差异分析，可以进行潜在致病因子的挖掘。离子交换富集法分别提取棉花组织诱导与非诱导体系中大丽轮枝菌分泌蛋白，利用iTRAQ标记和质谱检测技术进行分泌蛋白差异分析。结果表明，棉花组织诱导体系中有502个蛋白被上/下调表达，变化倍数在1.5倍以上，其中266个蛋白被注释为潜在致病因子，包括221个预测的分泌蛋白、107个细胞壁水解酶类蛋白、86个宿主-病原菌互作（PHI）蛋白、17个小的富含半胱氨酸蛋白、2个乙烯诱导坏死蛋白和1个赖氨酸模体（LysM）蛋白。另外，97个分泌型细胞壁水解酶类蛋白显著上调，它们主要参与果胶、纤维素和木质素的降解。实时定量PCR对显著上调的细胞壁水解酶进行体内表达水平检测，结果发现它们在大丽轮枝菌侵染棉花前和侵染后期基因低水平表达，在侵染的初期阶段均被显著诱导表达。可见，细胞壁水解酶在大丽轮枝菌侵染寄主过程中可能发挥着重要作用。比较基因组分析发现大丽轮枝菌参与果胶降解机制的酶类相比其它病原菌发生了明显扩增，尤其是GH28、GH43、PL1和PL3超家族成员。分泌蛋白谱鉴定结果中也发现了多个GH28、GH43、PL1和PL3超家族成员。其中，3个果胶裂解酶（PL3）超家族成员被显著上调，其上调倍数分别为15.02±0.26，13.17±0.92和7.01±0.8。构建PL3超家族进化树分析发现，VDAG₃551位于一个独立的进化分支，其它PL3超家族成员位于另一个进化分支。将VDAG₃551（VdPL3.1）编码基因敲除后，其突变体（ΔVdPL3.1）接种感病棉种的致病力明显下降，从野生型61%下降到29.3%。将VDAG₉536（VdPL3.3）编码基因敲除后，其突变体（ΔVdPL3.3）接种感病棉种的致病力为54.5%，相比野生型几乎没有改变。综上所述，本研究利用离子交换法对大丽轮枝菌VdG1在诱导和非诱导条件下的分泌蛋白进行提取，通过iTRAQ标记和质谱鉴定技术对分泌蛋白组差异蛋白进行定量分析，挖掘差异蛋白中潜在致病因子，分析大丽轮枝菌对棉花细胞壁组织的降解机制，验证了细胞壁水解酶PL3家族在大丽轮枝菌致病过程中的关键作用。更多还原

【Abstract】 To study the growth, generation of conidium and pathogenicity for secretome of V. dahliae andwhcich are influenced with natural sources, this fungus was cultured in the presence of cotton tissueextracts to mimic in vivo cotton infection system. The results showed that V. dahliae growth wassignificantly favored in the presence of host extracts, exhibiting the production of more spores andlarger amount of secretome compared with basic medium cultivation. Especially, the secretome inducedby cotton tissue caused more serious wilt phenotype in cotton leaves. To further identify theup-regulated secreted proteins induced by cotton tissue extract, we conducted an itraq-labeledcomparative proteomics of V. dahliae secretomes between the cotton tissue induction or non-inductionsamples. In total,502differentially expressed proteins were identified (Fold change>1.5or <0.6,p-value<0.05). Of which, a large number of potential pathogenicity factors were identified, including221secreted proteins,107plant cell wall degradation enzyme (PCWDE),86pathogen-host interaction(PHI) candidates,17small cysteine-rich proteins,2necrosis and ethylene-inducing-like proteins (NLPs)and a LysM effector candidate. Meanwhlie, we found that86secreted proteins were strongly induced(fold change>5.0, p-value<0.05), and76of which were PCWDEs which are suggested to involve inplant cell wall degradation and contribute to virulence in many fungal pathogens. To verify the inducedproteins exactly involved in cotton-V. dahliae interaction, we randomly selected some PCWDE genesand examined their transcripts by real-time quantitative PCR. The results revealed that most of selectedPCWDEs were activated in V. dahliae-cotton interaction, especially at the early of infection process. Inaddition, two of pectin lyses (PL) genes (VdPL3.1and VdPL3.3) belonging to PL3subfamily (there are11PL3genes in the V. dahliae genome) were further selected for knock-out assays. The resultsindicated that deletion of VdPL3.1, which separated from other PL3proteins in phylogenetically, causedover50%virulence reduction. However, loss-of-function of VdPL3.3did not affect virulence. Thatwould be caused by functional redundance among VdPL3.3and other PL3proteins in the samephylogenetic clade. This study provides a comprehensive resource for pathogenicity factor discovery inV. dahliae, and the highly induced PCWDEs are suggested to play significant roles in pathogenicity atthe early of cotton infection process. Further functional characterization of these PCWDEs may greatlyenhance the research on molecular mechanisms of pathogenicity in V. dahliae.更多还原