| The abalone Haliotis divericolor is a very important economic species in southern region of Fujian province.There will be significant academic value and economic importance if we conduct in-deep study on the mechanism of attachment and metamorphosis in this species.The process of attachment and metamorphosis is a very crucial biology phenomenon during the development of benthonic organisms,which holds the vital mechanism of adaptation in these organisms.We can learn further about the mechanism of attachment and metamorphosis,and exploit and utilize the related gene resources and find out the approach to improve the larval attachment rate and survival rate in shellfish culture by exploring some of the hidden mechanisms.In our earlier research,we found two gene families related to the attachment and metamorphosis process of H.diversicolor,named SARP19 gene family and vdg3 gene family.The two gene families highly expressed in digestive gland of H.diversicolor larvae at the metamorphosis stage.However,functional studies on these two gene families have not been reported.In this study,we chose two genes named SARP19-I1 and vdg3-Il,which were at the highest expression levels at the metamorphosis stage of H.diversicolor larvae from the two gene families SARP19 and vdg3 respectively,to construct yeast expression vector in Pichia pastoris.In order to obtain higher expression level of the target proteins,we chose the method of high density expression in fermentation tank other than the traditional shake flask cultivations to express the two target proteins in large-scale.We used circular dichroism spectrum to detect the secondary structure of the product to insure the structural integrity of the two recombinant proteins.After obtaining integral and pure recombinant proteins,we conducted in vitro activity trials on the two recombinant proteins and their mixture.We found that the recombinant proteins showed certain inhibition effect on the benthic diatom Nitzschia closterium f.minutissima.We also found that the recombinant proteins were cooperated with each other in the activity trial.We first expressed the recombinant proteins in the P.pastoris expression system using shake flasks with the volume of 1 L to cultivate the yeast.We obtained soluble SARP19-I1 and VDG3-I1 recombinant proteins in the supernatant of the expression product.After purifying the raw recombinant proteins,we obtained 4.49 mg/L SARP19-I1 recombinant proteins and 2.82 mg/L VDG3-I1 recombinant proteins.In order to obtain higher yield of the target proteins,we changed the expression method to the high density expression in fermentation tank and the yield of the recombinant proteins increased to 17.59 mg/L and 19.81 mg/L respectively,which was sufficient for the successive activities research.We conducted in vitro activity trials on the SARP 19-I1 protein,VDG3-I1 protein and their mixture SARP19-VDG3-I1,in order to explore their bioactivity function and synergism.The activity trials included bacteriostatic activity test and algal inhibition activity test.The results showed that the recombinant proteins and their mixture have certain algal inhibition activity on the diatom N.closterium f.minutissima.We investigated the action time,mixing ratio and active concentration of the recombinant proteins in algal inhibition activity test by using flow cytometer.The results indicated that the inhibition activity of SARP19-I1 protein varied with time,which reached the highest after reacting for 24 h.The inhibition activity of SARP19-I1 protein also varied with protein concentration in certain ranges,which reached the highest at 200?g/mL,but the activity was inhibited when the protein concentration was greater than 200 ?g/mL.The VDG3-I1 protein didn't show distinct inhibition to the diatom,while it showed inhibition activity immediately when mixed with small amount of SARP 19-I1 proteins,and the inhibition rate increased with the proportion of SARP 19-I1.In this study,we used the P.pastor is expression system to express the two genes SARP 19-11 and VDG3-I1,which were closely related to the metamorphosis of the H.diversicolor.We also conducted several trails in order to explore the bioactivities of the recombinant proteins,aiming at investigating the protein function of the two gene families and excavating the related upstream regulatory factors.We hope this study can expand the understanding of the mechanism of attachment and metamomhnsis as a hasio research and also can provide valuable ideas for exploitation of the related gene resources. |
PDF Full Text Request