The Study Of Mitochondrial Genome Of Ovomermis Sinensis And Romanomermis Wuchangensis

The mermithidae,a group of insect parasitic nematode,is a kind of precious host specificity and biological control resource which has very important value in research and practice in the insect comprehensive treatment in the field of agricultural,forestry and sanitation,the environment protecting,and the realization of diversified farmland creature.The mitochondrial genome,a main cellular organelle for oxidation of the most eukaryotes,is used widely as a molecular marker in the fields of systematic evolution, biogeography,and population genetics.Currently,the abroad study of this field basically focuses on the mitochondrial complete genome of six kinds of mermithidae(R.culicivorax,R.iyengari,R.nielseni,T. cosgrovci,H.agrotis,Agamermis sp.),whereas the domestic research is still on the way. In order to unveil the study of mermithidae mitochondrial complete genome in China in all directions,this thesis firstly makes a research on the mitochondrial genome of O. sinensis and R.wuchangensis in China.All the details are displayed in the following.1,A new method on the study of mitochondrial complete genome is established by combining the rolling circle amplification and two-step Long-PCR.The first step is to test the polymorphism of the nematode by rolling circle amplification;to cut the individual nematode which has phenomenon of polymorphism into two parts.The one is one-third part,and the other is two-thirds.The size of the nematode is confirmed by using of one-third part through the test of rolling circle amplification,and the sequence is gained by using of two-thirds part through the experiment of Long-PCR.The region should avoid repeated genes as much as possible during the design of the primers. Complete mitochondrial genome is achieved by the combination of two amplified fragments by Long-PCR,and the full sequence is fulfilled by the direct walking method. This method not only can elicit the size of complete mitochondrial genome in an accurate way and rapidly amplify the full sequence,but also can avoid tedious works as stitching many fragments and non-specific amplification in the region design of repeated genes. Hence it is considered as a simple,rapid and efficient method in the study of abundant repeated genes of mitochondrial genome.As to those mitochondrial genomes of simple nematode which is not with specialties in polymorphism,a way of directly extraction of genomic DNA by Long-PCR,and the amplification of full sequence by two-steps Long-PCR is advised.2,The mitochondrial genome of O.sinensis in this paper is studied in the above way. The result of rolling circle amplification shows that the mitochondrial genome of O. sinensis existing the phenomenon of polymorphism,ranging from13kb to 30kb in size. As to the materials of this experiment,there is no similarity in haplotype among all the 3 nematodes in this study.Two large fragments of 7kb and 12kb are amplified by two-step Long-PCR.Complete nucleotide sequences are determined by combining,the size is 18864bp.The base composition of the four nucleotides is A7498,T7217,C2077 and G2070, A%＞T%＞C%＞G%,T%+A%(78.02%)＞C%+G%(21.98%),showing a strong A+T bias.It contains genes of 15(12 species)proteins,2 ribosomal RNAs,22 transfer RNAs and a control region(D-loop).3,The research method of the mitochondrial genome of R.wuchangensis is the same as the study of mitochondrial genome of O.sinensis.The result of rolling circle amplification shows that the sizes of mitochondrial genome are the same in every individual nematode,without the phenomenon of polymorphism.Two large fragments of 5kb and 10kb are amplified by two-step Long-PCR,speculating its complete mitochondrial genome is about 15kb.At present,the sequence of the fragment 5kb has achieved while the fragment 10kb has sequenced 4kb.The sequence we have achieved is 8445bp in size,The base composition for the four nucleotides is A3824,T3088,C811and G721,A%:45.28,T%:36.57,G%:8.54,C%.9.60,containing genes of 7 proteins, 2 ribosomal RNAs,and 2 transfer RNAs.4,The phylogenetic trees are constructed for six species of mermithidae downloaded from GenBank and two species of mermithidae sequenced in this paper by adopting ATPase6 and COXⅡgenes sequences and their combined data.The result shows that four Romanomermis cluster together into one group.And after H.agrotis; Agamermis sp.has clustered together,O.sinensis begin to gather with them and form the second group.Finally two groups cluster with T.cosgrovci.By adopting the improved combination method of rolling circle amplification and two-step Long-PCR,this paper proves O.sinensis has the phenomenon of polymorphism and the R.wuchangensis doesn't.And complete mitochondrial genome of the O.sinensis and partial mitochondrial genome of the R.wuchangensis are achieved by amplification. This research can provides a resource for comparative mitochondrial genomics and systematic studies of other mermithidae nematodes.