| White spot syndrome virus(WSSV)is a highly lethal virus in prawn.The global prawn farming industry had lost $15 billion since the outbreak,and there is no effective way to control the virus.In laboratory,the main membrane protein VP28 of WSSV had been transferred into cyanobacteria and successfully expressed.The experimental results show that the transgenic algae can effectively protect prawn from the harm of WSSV.In this dissertation,another major membrane protein VP19 and fusion protein VP(19 + 28)on WSSV were transferred to Synechococcus sp.PCC7942 to study the protective effect of trans-vp19 and vp(19 + 28)genotypes of cyanobacteria on prawn seedlings,and the immune mechanism was preliminarily explored.The main results are as follows:(1)Construction and screening of Synechococcus sp.PCC 7942 harboring vp19 and vp(19 + 28)gene WSSV used as template,full-length primers were designed and the target gene fragments were obtained by PCR amplification.After result were sequenced correctly,the target gene fragments were transferred into plasmid p RL-489.p RL-489 plasmid which containing the target gene fragment was mixed with shuttle plasmid p RL-542 and wild type Synechococcus sp.PCC 7942.Then the mixture was coated on the BG-11(+N)plate through three-parent conjugation.The grown algae were transferred to a plate containing kanamycin antibiotic for solid plate screening,and then transferred to a liquid culture medium containing kanamycin antibiotic for gradient screening.(2)Study on the expression efficiency and photosynthetic physiological characteristics of Synechococcus sp.PCC 7942 harboring vp19 and vp(19 + 28)RT-PCR and Western Blot were used to detect Synechococcus sp.PCC 7942 harboring vp19 and vp(19 + 28),and the results showed that both vp19 and vp(19 +28)were successfully expressed at the molecular and protein levels.In order to improve the growth efficiency and target gene expression efficiency of transgenic algae,liquid oxygen electrodes were used to determine the optimal growth conditions of Synechococcus sp.PCC 7942 harboring vp19 and vp(19 + 28),and results were:temperature 38 ℃,light intensity 2 500 lx,p H 8.0,salinity 0 mol/L Na Cl.Results showed that harvest days were the key factor of expression rate.The expression rate of Synechococcus sp.PCC 7942 harboring vp19 at the later stage of logarithmic growth was the highest,reaching 9.2 %,and the protein yield of vp19 reached 7.3 mg/L;The expression rate of Synechococcus sp.PCC 7942 harboring vp(19 + 28)at the early stage of logarithmic growth was the highes,treaching 7.6 % and the yield of vp(19 + 28)reached 6.9 mg/L.(3)Study on the efficacy of Synechococcus sp.PCC 7942 harboring vp19,vp28 and vp(19 + 28)feeding Litopenaeus vannamei and seedling WSSV challenge experiment The results of WSSV challenge test showed that the Synechococcus sp.PCC 7942 harboring vp19,vp28,and vp(19 + 28)could improve the survival rate of L.vannamei and increase its growth rate,which is a strong proof of ‘both drug and food from the same feedstock’.Vp(19 + 28)group showed the highest survival rate compared with vp19 and vp28 groups,and survival rates increased by 8.9 % and13.3 %,respectively;protection rate against WSSV of vp19 group is lower than that of vp28 group,but the death trend of vp19 group is slower than that of vp28 group.Therefore,we speculate that bivalent VP(19 + 28)has a better protection rate than that of VP19 and VP28,and long-term protection rate of VP19 is better than that of VP28.The results of enzyme activity test showed that after WSSV infection,the final PO enzyme activity of the test group was significantly higher than that of the positive control group(P < 0.05).SOD activity decreased significantly in the WSSV group within 8 hours,and was significantly higher in the test group than the positive group at 48 hours(P < 0.05).The change of CAT activity was consistent with SOD activity.Finally,the enzyme activity of the experimental group was significantly higher than that of the positive control group(P < 0.05).LYZ activity in the WSSV group increased and then decreased with the change of infection time,and LYZ activity in each group was significantly lower than that in the negative control group after 72 h(P < 0.05).The result proves that Synechococcus harboring vp19,vp28,and vp(19 + 28)genes enhanced the activity of immune-related enzymes,and we speculated that oral Synechococcus mutants could enhance the growth performance,antioxidant capacity and furthermore reduce oxidative stress and immune depression challenged by WSSV.In addition,this study further explored the optimal growth conditions of transgenic algae,and explored the influence of different environmental factors on the growth of Synechococcus,so as to improve the growth activity of transgenic Synechococcus,and lay a foundation for the subculture of algae.In addition,RT-PCR was used to detect the expression of target genes in different growth stages of Synechococcus,so as to determine the optimal harvest time of transgenic algae,avoid blind collection,save the cost of culture,and provide a basis for the dosage of transgenic algae used in WSSV challenged experiment. |
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