辣椒N基因介导抗根结线虫作用早期表达基因的抑制性消减杂交SSH分析

Analysis of Early Expression Genes Resistance to Root Knot Nematode in N Gene Pepper by SSH

以含N基因的辣椒(Capsicum annuumL.)为试验材料,接种南方根结线虫(Meloidogyne in-cognita)12、24、36h的根尖材料作为测验方(tester),相应的未接种根尖材料作为驱动方(driver),构建一个南方根结线虫诱导N基因表达早期的正向抑制消减杂交cDNA文库,并结合文库高密度点阵膜杂交差异筛选,获得了237条表达序列标签(EST)。在GenBank上进行BLASTn与BLASTx分析,得到148条功能已知的EST序列,获得已知的上调抗性相关EST68个。分离出了具有NBS-LRR结构的抗线虫蛋白和类LRR抗性蛋白的基因,防御作用相关的类萌芽素(GLP)、HSR203J蛋白、蜜腺蛋白、蛇毒素肽等基因,抗性相关的WRKY、ERFBP等转录因子基因,以及G蛋白、14-3-3蛋白等多种信号蛋白基因。通过Gene Ontology分析,N基因介导的早期表达抗病基因涉及病原物的识别、抗性信号传导、过敏性坏死、系统获得性抗性以及植物细胞保护机制等多个方面,并有许多功能未知的基因有待于进一步的研究。

The nematode resistant gene N is a single dominant gene in pepper cuhivar Carolina Wonder （Capsicum annuum L. ）. To investigate the early up-regulated expression genes profile in cuhivar Carolina Wonder induced by root-knot nematode （Meloidogyne incognita, RKN）, a forward subtracted cDNA library was constructed using suppression subtractive hybridization （SSH）. The cDNA of pepper seedling root tips inoculated with J2 RKN for 12, 24 and 36 h were used as tester and that from untreated root tips as driver. Totally 237 RKN-induced SSH cDNA fragments were selected with DIG Nonradioactive Nucleic Acid Labeling and Detection System. Based on the sequencing and BLAST analyzing of 148 fragments, 68 up-regulated expression EST fragments were identified, including the genes encode nucleotide-binding site/leucine-rich repeat （NBS-LRR） disease resistant proteins and LRR resistance protein-like; some defense proteins, such as Germin-like protein, HSR203J protein-like, Nectarin, and Snakin-2; some biostress resistance related transcription factor like WRKY and ERFBP; some signal proteins, such as GRP-like protein and 14-3-3 family protein. The Gene Ontology analysis revealed that the up-regulated ESTs involved in the recognition to nematode, signal transduction, protection mechanism, HR and SAR. In addition, some ESTs of unknown function may be novel genes.