The Prokaryotic Expression And Protein Expression Analysis Of Oslaf-1from Ovomermis Sinensis

The insect-parasitic nematode, Ovomermis sinensis has potential for the biological control of a range of insect pests. O. sinensis absorbs nutrition from the host’s haemolymph only in the parasitic stages and their sexual determination is related with the quantity of the nutrition during parasitic stage. At present, we know little about the reproductive and developmental mechanism of O. sinensis, especially in the molecular biology. Therefore, it is important to research the related genes.DEAD-box proteins were found in bacteria, yeast, plant and animals, which was a kind of RNA helicase. They prevented RNA mis-folding and catalytic conformational change by using RNA-dependent ATPase activity. They played an important role in the RNA metabolic process. The study in animals found that they participate in many life processes, such as gonads development, the formation of germ cells, spermatogenesis, embryos development and organs differentiation, so they had a wide range of physiological significance.Previous works in our lab had obtained the Oslaf-1full cDNA in O. sinensis. Evolutionary analysis showed that the gene belonged to PL10subfamily in DEAD-box family. According to the expression pattern of Oslaf-1gene at different development stages and in different tissues of O. sinensis, we inferred that Oslaf-1gene was related with the gonadal development and function. The whole-mount in situ hybridization found that the gene transcripts was present widely in early embryos stage, and then the signal was mainly concentrated in the reproductive primordial in infective juveniles. The result demonstrated that the gene took part in the development of gonads and embryo. Previous studies were at the transcriptional level, and protein is a bearer in the cell metabolism process. The study carried out at the protein level, we tried to understand the role of Oslaf-1in the gonadal development and maturation of O. sinensis.We amplified the parts of Oslaf-1by PCR, which was1086bp. After confirmed by sequencing, it was connected with the expression vector; the recombinant vector was named as pMAL-c2X-Oslaf-1. Then, the pMAL-c2X-Oslaf-1was introduced into the E.coli BL21(DE3). The fusion protein was induced by IPTG and was identified by Western-blotting with MBP-tag antibody. The result showed that the fusion protein contain MBP-tag. After sonication, solubility of the fusion protein was checked by Western-blotting, showed that recombinant protein was mainly concentrated in the precipitation. Then, protein derived from cutting SDS-PAGE was used to immunize the BALB/c mice to produce an antibody against OSLAF-1. By Western-blotting analysis, we found that the antiserum could identify fusion protein in the recombinant bacteria, specifically.The OSLAF-1in different parts of O. sinensis was detected using the antiserum. The result showed that there was two black bands, the size was consistent with the predicted molecular weight (82.2KD). The protein was expressed in head, tail, testis and ovary, whereas low or no expression in the integument; the signal in the testis and ovary was significantly higher than in the head and tail (P<0.05), but there was not significant difference between the testis and ovary (P>0.05). This indicates that the protein maybe associated with the function of gonads.The research showed that it took about25d from last molting to adult, and the stage was a critical period of gonads development in O. sinensis. The OSLAF-1was expressed in the period of females nematode; and with gonads development, the relative expression level of OSLAF-1was increased, then reduced. We speculated it possible involved in the development and maturation of gonads in females; Compared with other days, the protein expression was highest at10and15days in post-parasitic nematode, and had significant difference with other days (for example0,20,25days of post-parasitic females nematode)(P<0.05), the quantity of OSLAF-1in5days of post-parasitic females nematode was not significant difference with any day (P>0.05). So we speculated10-15days in the post-parasitic stage maybe an important period that OSLAF-1work in female nematodes.