Isolation And Identification Of Parasitism Related Genes Form Meloidogyne Incognita And Rna Interference Research

Plant parasitic nematodes are economically important pests for many crops worldwide. Three groups of endoparasitic sedentary nematodes which belong to genera Globodera, Heterodera (cyst nematodes) and Meloidogyne (root-knot nematodes, RKN), cause enormous yield losses to world agriculture estimated at US$100billion annually. Among them, the root knot nematode Meloidogyne incognita, is considered to be the most dangerous pest because of its wide geographic distribution and host range. The second stage juveniles of root knot nematode enter the plant closely behind the root tip and migrate intercellularly. Subsequently they go to the vascular cylinder where they induce a permanent feeding site, giant-cell. The juveniles moult three times to adult female. Stylet secretions originated from esophageal gland cells of root knot nematodes are thought to play an important role in the interaction of these parasites with their host plants, therefore the cloning and identification of parasitism related genes encoding esophageal gland proteins secreted from the nematode stylet into host tissues is the key to understanding the molecular basis on parasitism of plants.Some pathogenicity related genes including cellulase, pectate lyase, expansin and chorismate mutase have been successfully cloned from some nematodes species using different molecular techniques. Nevertheless, the obligate biotrophic nature and refractory to mutation and transformation allow the functional analysis of genes more difficult. The development of RNA interference (RNAi) technology provides an effective tool for understanding gene function in plant parasitic nematodes and a new transgenetic strategy for controlling root-knot nematodes.In this study, the parasitism related genes from M. incognita were isolated by representational difference analysis (RDA); one isolated cDNA fragment which deduced amino acid sequences similar to venom allergen-like protein, its full length cDNA was cloned by rapid amplification of cDNA ends (RACE) and analyzed;, in vitro and in vivo RNAi research was carried out to validate the function of the isolated gene in parasitism of M. incognita. A representational difference analysis (RDA)-based strategy was used to identify parasitism related genes differentially expressed in two different parasitic stages of root knot nematode M. incognita, early parasitic second-stage juveniles and parasitic-stage juveniles. The DNA fragments derived from green fluorescent protein(gfp) were used as internal standards to monitor the effect of subtractive hybridization. Total of15cDNA fragments were screened and identified. Five out of15cDNA fragments were proved to be differentially expressed by reverse transcription-polymerase chain reaction (RT-PCR). Two cDNA fragments were found to be presented in the early parasitic second-stage juveniles and absented in the parasitic-stage juveniles, two were up-regulating expressed in the early parasitic second-stage juveniles and one was up-regulating expressed in parasitic-stage juveniles. In BLASTX searching and analysis, the sequences of5cDNA fragments did not show any significant similarity with sequences from known proteins, while the rest of ten were highly matched with sequences of reported proteins from phytoparasitic nematodes, free-living nematode Caenorhabditis elegans and animal parasitic nematodes.According to the cDNA sequences of B1b fragment which was derived from the forward RDA, the gene specific primers were designed. A venom allergen-like protein gene from M. incognita (designated Mi-vap-2) was successfully cloned by RACE and genomic amplification. The gene Mi-vap-2is1917bp long, contains three introns which range in size from39to797bp and four exons range in size from37to361bp. The cDNA of Mi-vap-2contains an open reading frame encoding294amino acids with the first16amino acids being a putative secretion signal. Southern blot analysis suggested that Mi-vp-2is probably the member of a small multigene family. In situ hybridization analyses showed the transcripts of Mi-vap-2accumulated exclusively within the subventral oesophageal gland cells of M. incognita. RT-PCR analyses confirmed Mi-vap-2was transcribed mainly in the pre-parasitic second-stage and early post-inoculation juveniles. All the results indicated that this venom allergen-like protein gene from M. incognita may play an important role in establishment of the parasitic relationship between plants and nematodes.The effectiveness of neurologically compounds of octopamine and resorcinol on stimulating the uptake of infective second-stage juveniles (J2) from M. incognita in vitro was compared. The results showed that octopamine didn’t have effect on stimulating the uptake of J2through stylet and pharynx, whereas resorcinol led to an apparently uptake. Optimization of the conditions for resorcinol to stimulate uptake in vitro by J2was carried out. cDNA of Mi-vap-2from M. incognita was used as template and in vitro transcription was performed to synthesize the VAP dsRNA. RNA interference(RNAi)mediated Mi-vap-2gene silencing was conducted based on the optimized conditions for uptaking. RT-PCR detections confirmed the decrease of Mi-vap-2transcription in nematode samples treated with VAP dsRNA. The reduction in the reproduction ability of nematodes was proved on sensitive tomato plants after dsRNA treatments. The successful RNAi in vitro for Mi-vap-2was demonstrated in this study which validated Mi-vap-2may have an essential function in parasitism of root-knot nematode to plants.The sense and anti-sense cDNA sequences of venom allergen-like protein gene Mi-vap-2from M. incognita were inserted into the vector pHANNIBAL. The transformed clone containing reverse repeat sequences was subcloned into the binary vector pART27and a dsRNA expression construct was produced. Subsequently the construct was introduced into tobacco by leaf-disk method with the help of Agrobacterium tumefaciens. Southern blot analysis showed that the construct was integrated into the genome of tobacco with single copy and obtained stable heredity in T2generation of transgenic lines. Northern blot analysis validated that the transgenic lines produced small interfering RNAs (siRNA) which was homologous to the Mi-vap-2. No obvious reduction in pathogenicity of M. incognita to transgenic tobacco plant was observed, which demonstrated that Mi-vap-2may be not an appropriated target gene for RNAi strategy of controlling root-knot nematodes. The strategy of silencing the target gene in root-knot nematode by expressing specific dsRNA in the host plant was proved to be practical in this study.