Cloning And Expression Of DEAD-box Gene And β-actin Gene From Two Species Of Mermithidae

Mermithidae,because of its enemy insect resources,potential of biocontrol and specific sexual differentiation mode which is determined by the nutrient competition during Parasitic stage,is becoming an ideal target for regulation studies to understand its sexual determination and sexual reproduction.DEAD-box proteins are the kinds of putative ATP-dependent RNA helicase,which can be found in becteria,yeast,plants and animals.Its activity is decided by 9 conserved domains.Till now there are 4 family members been reported—VASA,PL10,p68 and eIF4A subfamilies which play important roles in the occurrence of germcell,gonad development,cell proliferation and organ differentiation.In Caenorhabdites elegans, there exists a protein named VBH-1(Vasa and Belle-like helicase) which belongs to the DEAD-box protein family showing the germ cell specific expression pattern.And it plays important part in reproductive development because it is required for the switch from teste to ovary in hermaphrodite.In this thesis,we first cloned the cDNA of the DEAD-box genes from Mermithidae. Romanomermis wuchangensis's partial sequence contained 371bp and the conserved DEAD-box motif.We obtained its full cDNA in Ovomermis sinensis.The gene is 2596bp in length,and contains an ORF of 2237bp which encoded 701 amino acids. Sequence analysis showed that the predicted amino acid sequence shared 80%similarity to C.elegans VBH-1,and they were gathered in one branch.The gene was named Osvbh-1.The real-time quantitative PCR analysis showed that it was decreased as the gonads matured,suggesting its relationship with germ cell development.On the basis of its expression pattern,we conjectured that the rapid development of gonads approximately occurred in the last molting of O.sinensis.β-actin is highly conserved among species,and expresses constantly independent of experimental conditions.So,it is often used as an internal standard in quantitative analysis of expression levels of other genes.RT-PCR and RACE(rapid amplification of cDNA Ends) methods were used to cloneβ-actin gene in Mermithidae.R.wuchangensis's partial sequence contained 768bp.The full length ofβ-actin gene cDNA in O.sinensis was cloned for the first time.It is 1,636 bp in length,including an ORF of 1,131bp encoding a protein of 255 amino acids,its 5'-UTR and 3'-UTR is 137 bp,367 bp in length respectively.The nucleotide sequences and the amino acid sequences were analyzed by related programs.The expression pattern ofβ-actin gene was also analyzed by RT-PCR.The identities of O.sinensisβ-actin are above 95%,compared with other β-actins.The phylogenetic analysis was consistent with the traditional Classified Methods,suggesting that the O.sinensisβ-actin belongs to theβ-actin family.The expression of the clonedβ-actin gene was also consistent well in the different developmental stages of O.sinensis.It indicated that theβ-actin gene of O.sinensis was highly conserved,and can be used for the molecular marker of the biological evolution.It is also a good referenced gene for semi-quantitative analysis.