Cloning Two New Genes Of β-1, 4-endoglucanase From Ditylenchus Destructor And Functional Analysis With RNA Interference

Sweet potato nematodes are economically important pest for sweet potato product in China. It cause enormous yield losses to agriculture is very great annually. The stylet secretions originated from esophageal gland cells of nematodes are thought to play an important role in the interaction of these parasites with their host plants. From now on, the cloning and isolation of parasitism genes encoding esophageal gland proteins secreted from the nematode stylet into host plants is the key to understanding the molecular basis on parasitism of plants. In this study, two newsβ-1, 4-endoglucanase gene were isolated from Ditylenchus destructor, and analysis the genes and putative protein information by bioinformatics method. At the same time, in vitro RNAi research was carried out to validate the function of the target genes in parasitism of D. destructor.Two new genes ofβ-1, 4-endoglucanase, named Dd-eng-1and Dd-eng-2 respectivly,were cloning from Ditylenchus destructor, by RT-PCR and RACE. The full length cDNA of Dd-eng-1 consists of 1640 bp, with a 1443 bp ORF encoding a 481 amino acid protein (GenBank acession No. FJ430142). The putative protein with theoretical molecular weight is 50.89kD and pI 6.94. The sequenceing analysis indicated that this gene is classified as a member of Cellulase (glycosyl hydrolase family 5, GHF5) and has a 19 amino acids long singal sequence at N terminal and a high similarity to a bacterial type cellulose-binding domainⅡat C terminal. Comparative analysis between cDNA and Genomic showed that Dd-eng-1 contain six exons and five introns. The full length cDNA of Dd-eng-2 consists of 1164 bp, with a 1014bp ORF encoding a 338 amino acid protein (GenBank acession No. FJ374266) .The putative protein with theoretical molecular weight is 37.38kD and pI 5.5. The sequenceing analysis indicated that this gene is also classified as a member of Cellulase (glycosyl hydrolase family 5, GHF5) and has a 16 amino acids long singal sequence at N terminal but there have not the bacterial type cellulose-binding domainⅡat C terminal. Dd-eng-2 of genomic was consisted of five exons and four introns. Southern blot analysis suggested that Dd-eng-1 and Dd-eng-2 are probably the member of a small multigene family. The Dd-eng-2 recombinant protein was expressed in E.coli and the expression molecular weight was consistent with the expected value. Phylogenic analysis suggested that Dd-eng-1and Dd-eng-2 had a strong homology to Bacillus.subtilis and Erwina carotovora, which may be indicative of an ancient horizontal gene transfer from bacteria .In situ hybridization analysis showed that the transcripts of Dd-eng-1 and Dd-eng-2 accumulated exclusively within the subventral oesophageal cell of D. destructor. RT-PCR analysis confirmed that Dd-eng-1 and Dd-eng-2 were transcribed mainly in the female stage, and expressed at a lower level in J2 than male stage. At the same time, the stylet secretions of D. destructor showed clear cellulose activity in CMC plate assay, whereas in the negative controls no CMC hydrolysis was detected. The similar activity was observed in total homogenates and Dd-eng-2 recombinant protein was expressed in E.coli. This demonstrated that D. destructor produces and secrets functional celluloses.The effectiveness of neurologically compounds of resorcinol on stimulating the uptake of infective of D. destructor in vitro was studied. The results showed that the 1% resorcinol solution have effect on stimulating the uptake of nematode through stylet. In vitro transcription was performed to synthesize the Dd-eng-dsRNA. RNAi mediated Dd-eng-1and Dd-eng-2 genes silencing were conducted based on the optimized conditions for uptaking. Real time PCR detections confirmed the decrease of Dd-eng-1and Dd-eng-2 transcription in nematode treated with Dd-eng dsRNA before 1day. The lowest level was showed the nematode treated with dsRNA before 5 days . But the Dd-eng-1and Dd-eng-2transcription recovered by 5 to 10days post treatment. The reduction in the Percentage of penetrating nematodes was proved on sensitive sweet potato past treatments. In short, the successful RNAi in vitro for Dd-eng-1and Dd-eng-2 were demonstrated in this study which validated Dd-eng-1and Dd-eng-2 may have an essential function in parasitism of nematode to host.