Isolation And Expression Analysis Of MiMEK2 And MiLIN45 Of Plantnematode Meloidogyne Incognita

Ras-MAPK is an important class of signaling pathways, it plays a key role in the southern root-knot nematode growth and development. The Ras/Raf/Mek/Erk highly conserved signal transduction pathway exists in all eukaryotic cells. The broad participation of cell signaling pathways is in growth, proliferation, differentiation, apoptosis and metastasis process. To deeply study MAPK function is of importance to prevent plant-parasitic nematodes from infecting plant. In this study, the MiMEK2 (Mek) and MiLIN45 (Raf) gene were cloned by RT-PCR, the expression of MiMEK2 and MiLIN45 in deferent developmental stages was analyzed by q-RT-PCR and further on MiMEK2 and MiLIN45 gene expression in yeast, which explored the signal transduction pathway in nematode feeding behavior of the role of parasite and provided important information for nematode resistance breeding.The results were as follows:1. The gene cloning and structure analysis of MiMEK2 and MiLIN45 Based on the genome sequence of Meloidogyne incognita, the full sequence of MiMEK2 and MiLIN45 gene cDNA were predicted and cloned by RT-PCR. Their lengths were 1241 bp and 2592 bp (GenBank accession No.GU949543, No.GU949544) and contained the complete open reading frame, which encoded 323 and 863 amino acid residues. MiMEK2 gene was 7497 bp, contained 7 exons and MiLIN45 gene was 4422 bp, contained 17 exons. The full sequence was analysed through the EBI Interpro database, and shown that they are typical of serine / threonine-specific protein kinase family. The two genes were analysed by the TMHMM program and it demonstrated that they did not contain transmembrane. Multiple sequence alignment indicates that the amino acid level with high similarity with C. elegans, M. arenaria, Drosophila melanogaster, C. briggsae, Brugia malayi, Rattus norvegicu and M. incognita.2. The expression analysis of MiMEK2 and MiLIN45 in different instars of M.incognita by using RT-PCR and q-RT-PCR Total RNA was isolated from eggs, J2, female of M.incognita. First stand cDNA was synthesized using transcriptase. The expression level of the target genes was quantified by q-RT-PCR with specific primers designed according to gene sequences of MiMEK2 and MiLIN45 with actin as a reference. The results showed that MiMEK2 transcript highly presented in the eggs, but in the J2 larvae was lower. However, MiLIN45 was up regulated in the female compared with that in the J2. The result suggested that MiMEK2 and MiLIN45 seemed to be differentially expressed at different developmental stages. The stage-specific expression patterns of these two genes may be related to different functions during the parasite life cycle.3. The yeast expression of MiMEK2 and MiLIN45 Pichia pastoris expression vector was established by restriction enzyme digestion and connection and the GS115 was chosen as the expression strain of Pichia pastoris. The yeast strain GS115-pPIC9K-M1 and GS115-pPIC9K-M2, in which the MiMEK2 and MiLIN45 expressed respectively, were obtained through electro oration, transformants phenotype and identification of Colony PCR. After expression of methanol induction and electrophoretic analysis of SDS-PAGE, MiMEK2 gene, which was 35 kD in size, had been expressed accurately. The results were consistent with the expectation, but the protein encoded by MiLIN45 was of 60 kD compared with 90 kD presumed, so there was a large gap between the results and the speculation. This may be due to the large sequence of MiLIN45 and the improper selection of yeast strain.