【作者】 陆静；

【导师】 王源超；

【作者基本信息】 南京农业大学 ， 植物病理学， 2012， 硕士

【摘要】 大豆疫霉属于卵菌门,在亲缘关系上与真菌相差甚远而与褐藻和硅藻比较接近。由于大豆疫霉在生物学特征以及发病机制等方面与真菌差异较大,所以多数杀真菌剂对其没有效果。此外,大豆疫霉基因组不稳定,容易发生基因突变,导致它能够较快地对杀菌剂产生抗药性,也能迅速克服寄主植物的抗病性。大豆疫霉等4种疫霉菌全基因组序列的公布和疫霉菌遗传转化体系的建立,为我们从分子水平上了解疫霉菌的致病机理并且寻找广谱高效的疫霉菌杀菌剂提供了良好的平台。本研究充分发挥生物信息学技术优势,通过对大豆疫霉中一个与致病性相关的MAPK PsSAK1沉默突变体的DGE表达谱分析,筛选到一批可能受PsSAK1调控的候选基因。选取3个bZIP转录因子,结合PEG介导的大豆疫霉瞬时沉默体系得到沉默突变体后研究大豆疫霉bZIP转录因子的基因功能,为了解大豆疫霉分子致病机理提供了参考。转录因子是直接调控基因表达和应答特定信号途径的关键因子,研究大豆疫霉转录因子能找到直接调控大豆疫霉致病性相关的生物学过程的关键基因。通过生物信息学分析,我们在大豆疫霉基因组中预测了17个bZIP转录因子,其编码的蛋白长度从168到1042个氨基酸不等。该家族成员序列之间相似性低,并且在基因组上分布分散,只有Ps128339和Ps128400成簇排列于scaffold4。通过在线工具MEME,我们预测了大豆疫霉bZIP转录因子家族5种可能的功能域,其中motifl存在于所有大豆疫霉bZIP转录因子中且具有bZIP转录因子连续亮氨酸间隔排列的特征序列。根据转录模式的不同我们将bZIP转录因子家族分为5类,大多数成员在游动孢子、休止孢、休止孢萌发以及侵染阶段上调表达,根据上述差异表达基因推测大豆疫霉bZIP转录因子与大豆疫霉游动孢子和休止孢的发育过程以及致病过程相关。本章生物信息学分析为后续bZIP转录因子功能研究提供了依据。PsSAK1是一个与游动孢子活力及致病性相关的MAPK,为找到可能在下游受其调控的作用元件,在第一章生物信息学分析的基础上,本章研究挑选了3个在PsSAK1沉默突变体休止孢萌发阶段以及侵染早期均下调的大豆疫霉bZIP转录因子进行具体的功能分析,其中PsBZP1全长1622bp,含有5个内含子,编码的蛋白含413个氨基酸,qRT-PCR验证发现该基因在大豆疫霉游动孢子、休止孢以及休止孢萌发阶段均上调表达。通过大豆疫霉瞬时转化体系将其沉默,得到2个沉默效率分别为70%和60%的突变体。通过表型分析发现,PsBZP1的沉默不影响游动孢子趋化性和菌丝生长速率,但对游动孢子的游动性及活力有严重影响。PsBZP1沉默突变体游动孢子不能正常螺旋式前进而原地转圈游动,游动异常比率为39.3%。PsBZP1沉默突变体和野生型在2小时后的平均休止率分别为27.5%和19%,平均萌发率分别为78%和54%,均显著提高。PsBZP1沉默突变体休止孢萌发的芽管畸形,芽管顶端短粗卷曲出现畸形分支,休止孢萌发的畸形率为72.3%。此外,PsBZP1沉默突变体侵染大豆感病寄主的叶片和黄化苗下胚轴时出现明显的褐色坏死斑,致病性降低。上述结果表明,PsBZP1是一个对大豆疫霉游动孢子生长发育和致病性有关键调控作用的因子。更多还原

【Abstract】 Phytophthora sojae is a kind of oomycete so different from fungi on phylogenetic relationships, biological characters and pathogenic mechanism that most fungicides are failed to cure the root rot disease. Besides, because of the genomic instability of P. sojae, the genes are easily to mutant, causing quickly resistance to fungicide. With the completion of genome sequencing of4kinds of Phytophthora, it is possible to research the molecular pathogenic process of P. sojae, helping develop a new method to prevent the root rot disease. In this thesis, bioinformatics analysis as well as PEG-mediated transform system provided an effect method to identify the downstream regulated genes of PsSAK1and their gene functions. It helps to know much about the molecular infection mechanism of P. sojaeTranscription factors (TFs) play a critical role in regulating gene expression and responding to specific signal pathway. To identify the key factor in pathegetic process of P. sojae, we predicted17bZIP TFs from the genome. The result of bioinformatics analysis shows that the sequences of bZIP TFs in P. sojae have low similarity and locates on different scaffolds in genome, except that PsI28339and PsI28400located on the scaffold4. It is predicted that there are5motifs in the bZIP TFs by online tool MEME. According to different transcript patterns, we separated the bZIP TFs into5classes, most of which express much higher in the zoospore, cyte, germination cyte and infection stages. According to the differentially expressed genes, we speculate that the bZIP TFs of P. sojae are related to not only growth and development of zoospores and cytes but also the infection of P. sojae. Bioinformatics analysis supplies the important clues of follow up learning of gene functions.PsSAKl, encoding a MAP kinase, is an important regulator of zoospore development and pathogenicity in P. sojae. To find the TF which is downstream regulated by PsSAKl, we choose3candidates that are down-regulated in the CY and IF1.5in the PsSAK1-silenced lines. PsBZPl is a bZIP TF with a full length of1622bp, containing5 introns, encoding a protein at413animo acids. PsBZP1is up-regulated in zoospore, cyte, germination cyte and infection stages. To elucidate the function, the expressions of3candidates were silenced by transience silencing of P. sojae. Compaired with the wild type, the silenced strains, which relevant expression level is30%and40%, showed quicker encystment and higher germination ratio about27.5%and78%respectively. The zoospores of PsBZP1-silenced mutants swam in circle instead of swimming straight ahead, and the abnormal ratio is39.3%. Moreover, the cytes of them germinated abnormal germ tubes, much shorter and curvier on the top of the tubes, at the ratio of72.3%.PsBZP1-silenced mutants were unable to infect neither the soybean leaves nor the etiolated seedlings. Our results indicate that PsBZP1is an important TF, which possibly is in the downstream of PsSAK1, regulating zoospore development and pathogenicity in P. sojae.更多还原