| Grouper is a kind of marine edible fish rich in various mineral elements and vitamins,which is deeply loved by consumers.It is also an important aquaculture fish in the southeast coast of China(Fujian,Guangdong,Hainan)and other regions.In recent years,in the process of artificial breeding grouper,it is deeply troubled by many diseases such as iridovirus disease,enteritis,parasitic disease,etc.,of which viral nerve necrosis(VNN)is the most serious.The disease not only damages the interests of farmers,but also greatly hinders the development of aquaculture in China.VNN mainly breaks out in the breeding period of marine fish,i.e.young fish and naive fish.VNN invades the fish body by pathogenic nerve necrosis virus(NNV),resulting in abnormal behavior,imbalance of movement,anorexia and hunger strike of diseased fish,and eventually death of diseased fish,thus the virus is a greatly harmful and high incidence viral disease.So far,no specific drugs,effective vaccines or better prevention and treatment methods have been found to prevent or treat the disease,so the farming of grouper is still greatly hindered.Therefore,this study used genetically modified microalgae mixed with marine fish feed to feed groupers to obtain continuous antigen immunity from the seedling stage to prevent viral neuronecrosis.This research has been carried out from the following aspects:Screening,purification,seed conservation and large-scale cultivation of biological bait;electric shock transformation method is used to transform the pMaa7IR/DGNNVIR expression vector into Chlorella,and the selection of transgenes is carried out through screening,DNA identification and other steps Chlorella.Firstly,the mixed feed made from Transgenic Chlorella vulgaris and sea fish feed was fed to grouper,and then the toxicity test was carried out,and the preventive effect was evaluated.The result is as follows:The first study:Chlorella was stored on a solid TAP medium by streaking on a plate,and expanded by a stepwise expansion method.After comparing open culture and photobioreactor culture,it is found that the use of photobioreactor to cultivate chlorella has a faster growth rate and a higher final density.Finally,1500L of chlorella solution was obtained by cultivating chlorella in a photobioreactor,and the chlorella obtained was used for subsequent temporary culture of grouper.The second study:Pure and pollution-free rotifers were cultured as biological bait in grouper culture.The culture conditions of "S" type Brachionus plicatum were determined from the aspects of salinity,pH and feed amount of culture water.The results of this study showed that when the salinity of aquaculture water was 20～30,the rotifer population density and daily average proliferation rate reached the maximum.When the salinity was 10～20,the population density increased with the increase of salinity.When the salinity was lower than 10,the rotifer proliferation efficiency gradually decreased with the decrease of salinity,and the rotifer died completely when the salinity was lower than 5.When pH was in the range of 7.5～8.5,the population density and daily average proliferation rate reached the maximum.Under the suitable conditions such as salinity,pH and temperature,the maximum population density could be obtained by maintaining the density of Chlorella in rotifer culture medium above 5×106/mL in each feeding gradient set in this experiment.Therefore,it is concluded that the shorter the feeding interval and the larger the feeding amount,the faster the growth rate of rotifer population is,and the finally achievable population density will also slightly increase.The third study:The recombinant vector pMaa7IR/DGNNVIR was transferred into Chlorella vulgaris by electrotransformation.The electric shock conditions were as follows:voltage 800V～1600V,pulse length 032s,pulse interval 200ms,pulse times 90,electric shock 2～3 times;solid TAP medium with 10g/mL paromycin was used for screening,and DNA identification was carried out on the screened Chlorella vulgaris.Finally,8 transgenic Chlorella vulgaris were obtained,and 1500L of transgenic Chlorella vulgaris solution was obtained through expanded culture of transgenic Chlorella vulgaris.The fourth study:The collected Chlorella algae liquid was centrifuged,the algae mud was scraped,and the mixed feed was prepared according to the ratio of algae mud to feed(1:1).Finally,950g of mixed feed particles were prepared.The grouper was continuously fed with mixed feed for 10 days to accept the virus attack experiment by intraperitoneal injection with virus extract(24 copies/μL)which has determined virus copies.Then,the effect was evaluated at molecular,cytological and biological level.The results showed that after 48 hours of attack,the virus content in the eyes and brain tissues of grouper in the experimental group decreased by 51.70%,48.47%and 53.02%respectively compared with that in the control group.After 48 hours of virus attack,tissue sections from eyes and brain were made and there were clearly vacuolized pathological changes in fish eye retina and brain tissues.On the 2nd,4th,6th,10th and 14th days after the attack,samples were taken from grouper in each group to determine the copy number of in vivo nerve necrosis virus.The results showed that the growth trend of virus in grouper in the experimental group was lower than that in the control group,and the copy number of virus was smaller than that in the control group.The final survival rate was counted 14 days after the attack,with 26.0%,22.0%and 26.0%higher in DGNNVRNAi1,DGNNVRNAi2 and DGNNVRNAi3 than that of the control group,respectively. |
