基于转录组测序的象耳豆根结线虫致病相关基因初步筛选

Preliminary screening of genes related to pathogenicity of Meloidogyne enterolobii based on transcriptome testing

[目的]象耳豆根结线虫具有致病力强、分布范围广、危害严重、且能克服多数栽培作物中的抗根结线虫基因抗性的特性,为了探究象耳豆根结线虫克服抗线虫基因抗性的机制,本研究以象耳豆根结线虫与携带Mi-1基因番茄的互作为研究对象。[方法]将象耳豆根结线虫接种番茄VFNT植株,运用转录组测序技术比较分析象耳豆根结线虫侵染番茄根系2~6 d关键时期基因转录情况,筛选差异表达基因并对上调基因进行功能和代谢通路的分析,再利用生物信息学软件筛选出具有信号肽且无跨膜结构域的象耳豆根结线虫候选致病相关基因,并进行qPCR试验验证。[结果]象耳豆根结线虫侵染后在侵染前期差异表达基因共计967个,其中上调基因647个;差异上调基因功能及代谢过程都有明显变化且多与侵染相关,通过生物信息学分析发现其中的29个基因符合线虫致病相关基因特征,结合基因序列比对及结构域分析从中挑选出信度最高的5个基因,qPCR验证上述5个基因均在象耳豆根结线虫寄生关键阶段特异性上调表达。[结论]通过象耳豆根结线虫侵染携带番茄VFNT植株转录组进行分析从而初步筛选致病相关基因,为进一步了解象耳豆根结线虫强致病机理和制定相关防治策略奠定了基础。

[Objective]Meloidogyne enterolobii has the characteristics of high pathogenicity, wide distribution, serious damage, and the ability to overcome the resistant genes to root-knot nematode in most cultivated crops. In order to explore the mechanism by which root knot nematode overcomes the resistance of the nematode-resistant gene, the interaction between M. enterolobii and tomato carrying Mi-1 gene was studied. [Methods]Root knot nematodes were inoculated to tomato VFNT plants, and transcriptome sequencing technology was used to compare and analyze gene transcription during the critical period of 2~6 days after the infestation of root-knot nematode to tomato roots. The differentially expressed genes were screened. The function and metabolic pathways of the upregulated genes were analyzed. The bioinformatics software was used to screen the candidate pathogenicity-related genes of root-knot nematode with signal peptides but no transmembrane domains, and qPCR experiments was performed to verify the identified genes.[Results]The results showed that a total of 967 differentially expressed genes were identified in the pre-infection period after inoculation, of which 647 genes were up-regulated. The function and metabolic process of differentially up-regulated genes displayed the significant changes, and most of them were related to the infection. Bioinformatics analysis results indicated that a total of 29 genes were found to be consistent with the characteristics of nematode pathogenicity related genes. Five genes with the highest reliability were selected through the gene sequence alignment and domain analysis, and all of them were verified to be specifically upregulated at the critical stage of parasitism of M. enterolobii by qPCR.[Conclusion]Through the analysis of transcriptome of tomato VFNT-carrying plants infected by M. enterolobii,the pathogenic genes were screened, which will lay a foundation for further understanding the strong pathogenic mechanism of M. enterolobii and formulating related control strategies.