Cloning And Identification Of β-1,4-endoglucanase Gene Of Rice Root Nematode Based On Transcriptome Analysis

Plant parasitic nematodes are one of the most important pathogens that harm rice and cause significant losses to rice.In the process of infecting host plants,nematode can degrade the plant cell wall by secreting cell wall degradation enzyme to help infect and complete the parasitic process.Among the large number of cell wall degradation enzymes secreted by nematodes,the β-1,4-endoglucanase is one of the most studied species at present.β-1,4-endoglycosylase can hydrolyze the 1,4 glycosidic bond in the plant cell wall,destroy the structure of cellulose,and eventually degrade the cell wall of the plant.To study the β-1,4-endoglucanase gene(Hm-eng-1)biological function in the infection process of rice root nematode(Hirschmanniella spp),the thesis is organized as follows:1.The analysis of the transcriptome data of H.mucronata: Analyzing the RNA-seq results of infected-rice nematode R51,R155 and non-infected-rice nematode NHP,when RPKM >0.3,there were about 143 thousand genes expressing in the samples,about 82.3% of the nematode genome encoding genes.The genes coding citric acid cycle,glycolytic pathway,RNA transport,amino acid synthesis,fatty acid metabolism,and cell wall degrading enzymes were up regulated.The results indicated that the nematode metabolism level has magnificently enhanced which should provide essentia basis for the nematode infection.2.Cloning and sequence analysis of Hm-eng-1 gene of rice root nematode: the full-length cDNA was obtained by RT-PCR and RACE.The new β-1,4-endoglucanase gene(Hm-eng-1,GenBank:MH203228)was clone.The cDNA of Hm-eng-1 is 1,035 bp and encodes 345 amino acids.There is a 21 aa signal peptide in the N-terminal.The amino acid sequence was found to belong to the fifth family of hydrolyzed glycosidase with a cellulose catalytic domain.The phylogenetic tree of ENGs protein of various parasitic nematodes were analyzed.It was shown that the HM-ENG-1 protein with HG-ENG-1,GR-ENG-1,GR-ENG-2 were all in the same evolutionary branch.3.The expression localization of Hm-eng-1: the single stranded DNA probes were prepared and studied by in situ hybridization.The results showed that Hm-eng-1 gene was specific expression in the subventral esophageal gland cell of rice root nematode,and its expression pattern is consistent with most eng genes of plant parasitic nematodes.4.The prokaryotic expression of the Hm-eng-1: the full length cDNA of Hm-eng-1 gene was cloned into prokaryotic expression vector pEASY-E1.Then the recombinant vector pEASY-E1-Hm-eng-1 was transformed to the host strain BL21(DE3).The expression recombinant was induced by IPTG.A protein about 38 KDa was found by SDS-PAGE and Western Blot analysis of the recimbinant Hm-eng-1.5.RNAi technology analysis Hm-eng-1 gene function: the specific segments of Hm-eng-1 were rearranged through Gateway technology to RNAi vector pB7 G,and then the recombinant interference vector pB7G-Hm-eng-1 was transfected into rice.The function of Hm-eng-1 gene was analyzed after successfully obtaining the the transgenic rice plant.