杂交竹梢枯病菌全基因组解析及致病关键基因功能验证

Bambusa pervariabilis × Dendrocalamopisis grandis is an important bamboo species of forest reclamation (from farmland) and paper timbers in Upper and Middle reaches of Changjiang River. But the extensive occurrence of bamboo blight disease seriously threatened the development. The protein toxin and action site of the patogen (Arthrinium phaeospermum) had been researched, but it has not referred to gene level, and the molecular basis of pathogenic mechanism has not quite clear. In the project, the genome DNA is extracted, the genome of A. phaeospermum is sequenced by the second-third joint assembly technology. The genome components including code area, noncoding RNA, repetitive sequence are analysed by diverse bioinformatics analysis softwares and online tools. The pathogenic genes including pathogen host interactions, trascription factors, secretory protein, secondary metabolic enzymes are annotated. After RNA-seq of different infection stages of hybrid bamboo twigs, the differences of gene expression are analysed, and the pathogenic genes are inferred. Based for this researches, the comfirmation of the key pathogenic genes is used by gene knockout by ATMT. The mutants' phenotypes are analysed by hyphal growth and sporulation capacity, spore germination and attachment spore formation, pathogenicity of potting bamboos and infection process microexamination. This reasearch will provide reliable theoretical basis for the intensive study of pathogenic mechanism and molecular mechanism of pathogen host interaction.

撑×绿杂交竹是长江中上游地区退耕还林和造纸用材的重要竹种，但近年来梢枯病的大面积发生严重威胁其发展。申请者已对引起该病害的病原—暗孢节菱孢菌进行了蛋白毒素及作用位点方面的研究，但并未涉及基因水平，对致病机理的分子基础不甚明了。本项目提取该菌基因组DNA，采用二/三代联合组装技术进行测序，利用生物信息学分析软件、在线工具进行编码区、非编码RNA、重复序列的基因组组分分析，病原与宿主互作因子、转录因子、分泌蛋白、次生代谢酶等致病基因注释。通过病菌侵染杂交竹嫩枝不同阶段的转录组测序，分析基因表达情况差异，从中推测与致病有关的基因。以此为基础，采用根癌农杆菌介导同源重组进行基因敲除的方法进行关键致病基因的验证，通过菌丝生长与产孢量、孢子萌发与附着胞形成、盆栽杂交竹致病性、侵染过程显微观察等对突变体进行表型分析。本研究旨在为杂交竹梢枯病致病机理及病原与寄主互作分子机制的深入研究提供可靠理论基础。

以暗孢节菱孢菌为对象，在研究其全基因组和不同培养条件下转录组基础上，对关键致病基因进行筛选、鉴定和功能验证分析。比较该病原在不同培养条件中差异表达基因，并通过PEG介导的原声质体基因敲除法分析该菌内部导致杂交竹梢枯病的关键致病基因和验证致病基因的功能,为该病致病机理及病原与寄主互作分子机制的深入研究提供可靠理论基础。获得以下主要研究成果：1. 采用高通量Illumina HiSeq 4000系统与PacBio RSII克隆测序平台相结合得到7.76Gb数据，总长度为48,449,939，GC含量53.05%。得到19,836个基因，结果显示有18071、11451、9091、15565、6559、1917、3611和9050个基因注释到NR、SwissProt、KOG、KEGG、GO、CAZy、PHI和Pfam数据库。样品基因组AP-Z13较FO-v2、AciTa-1、UCR-E1和RN-2四个参考基因组的共线性分析中分别有1746、786、1957和1673个共线性区块。2. 对经无菌去离子水(CK)、无菌水稻组织液(T1)和无菌杂交竹组织液(T2)培养基培养后的菌株AP-Z13进行转录组测序，分别得到12845、12697和13055个基因，在Swissport、KEGG、KOG和NR四大数据库中分别注释到7271、3714、5904和9991个基因。CK与T1的差异分析中有4个差异基因上调，367个差异基因下调；CK与T2的差异分析中有263个差异基因上调，161个基因下调，T1和T2的差异分析中，有323个差异基因上调，148个基因下调。荧光定量PCR结果显示，从CK-T2、T-T2和CK-T1处理组中分别有9、9和8个基因的变化趋势与转录组测序结果一致，结果一致率超过80%，结果可靠。3. Lysing和Driselease的用量分别为0.2g和0.5g，溶解于20mL 1.2M的KCl缓冲液得到的酶解液酶解菌丝效果较好，菌丝的量为100mL PDB培养基中接种直径为5mm菌饼28，180r/min摇培48h。最终成功构建敲除载体0193、0194、2252、2267和2268共5个敲除载体，通过PEG介导原生质体进行遗传转化后得到0193，0194和2252三个基因缺失体转化子。0193缺失体转化子较野生型菌株生长较慢，孢子量更少，致病性降低。