乳酸乳球菌对货架期杏鲍菇致病相关基因的鉴定及功能解析

In recent years, the industrial cultivation of an edible basidiomycete Pleurotus eryngii is rapidly expanding in China. However, the healthy development of this industry is seriously affected by the postharvest decay of P. eryngii. Recently, the applicant found that Lactococcus lactis subsp. lactis was a major cause of the decay of postharvest P. eryngii, and preliminarily analyzed its pathogenesis. Based on the previous research, this study plans to construct the mutant library of L. lactis subsp. lactis strain SLPE1-3 by EZ-Tn5 transposon, screen mutants which completely or partly lose pathogenicity to postharvest P. eryngii, and identify and clone 3-5 potentially pathogenicity-related genes. Then, in-frame deletion mutants of these genes are constructed by pBSⅡ-KS+ vector, whose pathogenictiy and other biological characteristics are determined. Two or three important pathogenicity-related genes will be obtained. At last, the transcriptional levels of pathogenicity-related genes are determined during the interaction between L. lactis subsp. lactis and P. eryngii by the qRT-PCR technique, and their biological functions are analyzed during the interaction by a scanning electron microscope. The successful implementation of this study will help to illuminate the pathogenesis mechanisms of L. lactis subsp. lactis, and provide theoretical foundations and new technical thinking for the preservation of postharvest P. eryngii.

近年来，杏鲍菇的工厂化栽培产业在我国迅速发展壮大，但是，货架期杏鲍菇的腐败变质严重制约了该产业的健康发展。最近，申请人发现乳酸乳球菌乳酸亚种的侵染是导致货架期杏鲍菇腐败变质的主要原因，并初步分析了其致病机理。在此基础上，本研究拟通过构建EZ-Tn5转座子插入突变体文库，筛选致病力丧失或明显减弱的突变体，鉴定并克隆3-5个候选致病相关基因；接着，利用pBSⅡ-KS+载体定点敲除这些候选基因，对缺失突变体的致病性等生物学表型进行测定，期望获得2-3个重要的致病相关基因；最后，借助qRT-PCR技术分析致病相关基因在乳酸乳球菌乳酸亚种对杏鲍菇侵染过程中的转录表达规律，利用扫描电镜观察分析致病相关基因在此侵染过程中的生物学功能。本研究的顺利实施将可以初步阐明乳酸乳球菌乳酸亚种对货架期杏鲍菇的侵染致病机制，为货架期杏鲍菇的防腐保鲜提供理论依据和新的技术思路。

乳酸乳球菌乳酸亚种引起的溃烂病是造成采后杏鲍菇腐烂变质的主要原因。目前，仅对该病害进行了简单报道。为了解析乳酸乳球菌乳酸亚种对货架期杏鲍菇的侵染致病机制，本项目实施过程中主要开展了以下工作：（1）进一步优化了乳酸乳球菌乳酸亚种对货架期杏鲍菇的致病性测定方法，使接种杏鲍菇的发病率达100%（第12 d），病情指数达7级（第12 d）；（2）建立了乳酸乳球菌乳酸亚种SLPE1-3菌株的EZ-Tn5转座子插入突变体文库，包含1252个转化子，但是与野生型相比它们的致病性均没有明显减弱，这表明SLPE1-3的致病性是由多个基因联合决定；（3）利用单分子实时测序技术获得了SLPE1-3菌株的精细基因组图谱，由1个环状染色体组成，长度为2,522,493 bp，GC含量34.91%，含有37个潜在抗真菌相关基因；（4）建立了SLPE1-3与杏鲍菇的稳定互作体系，利用扫描电镜观察两者的互作和侵染过程，结果发现，在对峙培养及侵染过程中，大量SLPE1-3菌体黏附甚至镶嵌到杏鲍菇菌丝体细胞壁上，导致菌丝体皱缩、干瘪、死亡；（5）利用GC-MS技术测定互作过程中的次生代谢产物，共计鉴定到69个差异次生代谢物，其中2-羟基-苯基丙酸（PLA）能够导致杏鲍菇菌丝体皱缩、干瘪、死亡；（6）利用RNA-sequence技术比较分析互作过成中SLPE1-3和杏鲍菇的基因表达水平，结果发现， SLPE1-3中59个基因上调表达，杏鲍菇中80个基因上调表达。上述研究结果初步揭示了乳酸乳球菌乳酸亚种对杏鲍菇的侵染致病机制，明确了PLA是乳酸乳球菌乳酸亚种侵染货架期杏鲍菇的关键致病因子，为采后杏鲍菇的防腐保鲜提供了理论和试验支持。项目执行期内，发表SCI论文4篇（标注）；申请国家发明专利3项，获得授权发明专利1项；培养研究生1名。本研究完成了项目规定的考核指标。