Cloning Of Meactin And MeHsp70Genes From Meloidogyne Enterolobii And MeHsp70Prokaryotic Expression

Root-knot nematodes (Meloidogyne spp.) are root endoparasites of plants, that are capable of causing considerable damage on a variety of crops. To date, approximately30species of Meloidogyne have been recorded in China, of which M. enterolobii has widespread distribution in Hainan Island, it is also found in Guangdong province. As the species identification of Root-knot nematode on melons vegetables, solanaceous vegetable and Na Yao plants in Hainan Island, found that the diffusion of M. enterolobii populations was serious. It is shown to replace Meloidogyne incognita, and become the most important root-knot nematodes in tropical and subtropical regions kinds of trend. In recent years, much attention has been paid to the study of M. enterolobii, however, little reports has addressed on the functional genes and functional genomics, molecular biology research in M. enterolobii. Study to M. enterolobii for material, the full length cDNA of Actin and Hsp70from M. enterolobii respectively ware cloned by using RT-PCR, rapid amplification of cDNA ends (RACE). Bioinformatics analysis and MeHsp70prokaryotic expression analysis were carried out. Now the major results studied in this paper are abstracted as follows:(1) Me Actin full-length cDNA sequence has1298bp, one complete ORF with1125bp, start codon at82site, stop codon at1207site, and encoding375amino acids(accession number: KF534787). Which is highly conservative with known Actin of other species. The M. enterolobii Actin protein sequence showed99.20%and98.67%identity to Ditylenchus destructorto and Bursaphelenchus xylophilus,98.67%and97.33%identity to Caenorhabditis elegans and Homo sapiens, respectively. The M. enterolobii Actin gene may be-Actin by the construction of the evolutionary tree. It is also indicated that this Actin is more similar to the human cytoplasmic Actin ($-Actin) than to human muscle Actin(a-Actin).(2) MeHsp70full-length cDNA sequence has2203bp containing an open reading frame (ORF) of1959bp encoding a polypeptide of653amino acids with a predicted molecular mass of71.09kDa, which carried three important and intact Hsp70signature sequences, which was registered in GenBank with accession No. KF739434. The result of sequence similarity-analysis revealed that the translated molecules showed high homology to other eukarya. The Hsp70s system of evolutionary tree can not adequately reflect the relationship between species, speculate that the Hsp70functional similarity to construct the evolutionary tree in different species. Bioinformatics analysis revealed that the protein encoded by Hsp70gene in M. enterolobii was hydrophilic in most region without signal peptide and notable transmembrane region which indicated that the protein was not secretory protein. Secondary structurs of the protein were mainly alpha helixes and random coils. The Hsp70of M. enterolobii also had helical segment. The complete ORF encoding MeHsp70was inserted into pEASY-E1and pET30a(+), expression vectors pEASY-E1-MeHsp70and pET30a-MeHsp70were constructed. The MeHsp70protein was induced to express by0.4-1.0mmol/LIPTG Recombinant MeHsp70was over expressed in E. coli to study its possible function under temperature stress. The result suggested that MeHsp70provided thermotolerance to the transformed E. coli.