The Identification Of RDNA-ITS And Localization Of Heterochronic Gene In Meloidogyne Javanica

Root-knot nematodes are important endo-parasites in economic plants. The roots of tomatoes infected with M. javanica were got from the field of Tong'an rural area and roots were dissected.The egg masses, pre-parasitic juveniles and female adults of M. javanica were collected in the lab.The embryo development and different stages of life cycle of root-knot nematode were observed. In order to obtain a large number of nematodes in the lab, the tomatoes were planted in the laboratory and infected with the L2 stage for the further study.The rDNA-ITS1 and rDNA-ITS2 of M. javanica from different hosts and M.incognita were amplified in this study .The rDNA-ITS1 sequences from Genebank were used to compare the sequences by software clustalX and the phylogenetic trees was constructed .The results showed that the rDNA-ITS1 sequences of M. javanica from tomato and taraxacum are very similar to them. There is only 1bp difference between them. The rDNA-ITS1 sequences of M. javanica and M.incognita have 187bp differences from each other. Comparing the genetic distance of the root-knot nematodes, the average genetic distance of the M. javanica among innerspecies is closer than that of interspecies (between M. javanica and M.incognita). It might inform that M. javanica from tomato and taraxacum have evolutionarily closer relationship than that between M. javanica and M.incognita. ITS sequence can be a useful method to identify different species of root-knot nematodes.Whole-mount in situ hybridization was used to detect the distribution of the heterochronic genes in the embryo, L1 and L2 stage of M. javanica. The heterochronic gene let-7 mRNA exists in the two poles of the embryo , epidermic cells of the embryo and also presents in the head,esophageal bulb,intestine and tail of the L1 and L2 stage of M. javanica. The distribution of lin-28 mRNA in the embryo is same as that of the let-7 mRNA . The lin-28 mRNA presents mostly in the head,intestine and tail. The distribution of the lin-28 mRNA in L2 stage is not as broad as it in L1 stage.It might have the similar mechanism which the expression of lin-28 in L2 stage of C.elegans is inhibited by lin-4 and lin-14.The distribution of the lin-4 mRNA is in the body wall of the early development of the juvenile. The lin-14 mRNA distributes in the cells of ectoderm of the embryo and body wall,esophageal bulb and intestine of L1 and L2 stage.The genomic DNA and cDNA of M. javanica were used as template, and specific primers were designed to amplify a homologus of C.elegans lin-28. Then the fragments were subcloned in the plasmid pMD18-T, and transformed into the bacteria DH5α. The plasmids were digested by the restrictive enzymes. The sequence of C.elegans lin-28 homologus in M. javanica is very similar to the sequence of C.elegans lin-28. After compare the sequence in the Genedoc software , the similarity of three sequences was about 83%. The results provided the basic information for the further study of the function of lin-28 homologous in M. javanica.