Transfer Gene Of NNVCP Into Chlamydomonas To Prevent Grouper Viral Nervous Necrosis

1.Conservation,isolation,purification and expansion of economic algae strainsThe liquid impure algae strains of six economic algae strains were separated by dilution separation method,and six kinds of economical algae strains were successfully separated and purified;the ammoniase was used at concentrations of 10,20,40,60,80 and 100 pg/ml.Penicillin was successfully subjected to plate scribing method to successfully purify salt algae,seawater chlorella,and cyanobacteria on a solid medium.The purified algae were separately seeded with the corresponding medium.The three kinds of economical algae strains were expanded in liquid state by stepwise enlargement culture method.The final volume of each expanded strain was 100 L.During the period,the total expansion was 5 times,and the total economical algae strain was 500 L.This experiment not only increases the type of laboratory strain of algae strain,but also expands the cultivated algae strain for the production practice of seed production,and realizes the value of production and research.2.Construction of recombinant vector,transformation of Chlamydomonas and screeningThe expression vector pCAMBIA1302-NNVCP was successfully constructed by NCBI searching for the NNVCP gene and pCAMBIA 1302 vector recombination.The pCAMBIA 1302-NN VCP eukaryotic expression vector was transferred into Chlamydomonas.Resistance screening of pCAMBIA1302-NNVCP transgenic algal strains.The results showed that the pCAMBIA 1302-NNVCP transgenic strains were screened by TAP solid medium containing hygromycin 2,5,10,15 ?g/ml,respectively.42 strains of successful algae were screened and finally inoculated with 10 ?g/ml tide.Solid TAP medium for mycin.3.Identification,conservation,expansion and harvesting of transgenic algae strainsThe transgenic algal strains after resistance screening were identified,subjected to DNA and RNA extraction,and subjected to PCR verification.The results showed that 26 of the pCAMBIA1302-NNVCP strains were positive.The obtained 26 transgenic algae strains were preserved in a plate medium containing the corresponding antibiotics,and the transgenic algae liquid expanded culture was carried out,and the masses of pCAMBIA 1302-NNVCP-14/15/16 algae liquid were expanded to be 60.2 g and 66.7 g,respectively.69.2g,65.7g,provide experimental materials for the prevention of grouper VNN by transgenic microalgae.4.Preliminary diagnosis of Hainan pearl gentian grouper VNNCollecting the diseased fry of the pearl gentian grouper cultured by a grouper breeding base in Ledong County,Hainan Province.Through diagnosis,the results showed that typical VNN symptoms appeared,and the parasitological examination of the diseased fish body was negative.The RT-PCR method and OIE recommended primers showed that the specific fragment size of the PCR product of VNN was 421 bp.The cytomegaly virus and iridescent virus detection primers were used,and the mixed infection was excluded by RT-PCR method,and the initial diagnosis was VNN.Prior to this,it was rarely reported that VNN occurred in the winter,so this study is of great significance for the in-depth exploration of VNN and provides materials for the challenge experiments.5.transgenic microalgae to prevent pearl gentian grouper VNN experimentExplore the production methods of two kinds of simple self-made genetically engineered microalgae mixed feed.After feeding the GM mixed feed for ten consecutive days,the challenge experiment was carried out.From the molecular level,cytological level,and biological level,the results showed that the VNN virus coat protein mRNA content in the experimental group decreased by 67.25%,36.61%,and 77.17%,respectively,after 24 hours of challenge;The number of vacuolated cells in the experimental group was less than that in the control group,and the vacuolar lesions were slightly milder than the control group.The average survival rate of the experimental group was 33.33%higher than that of the control group,indicating that the NNVCP gene was resistant to the grouper virus.Necrosis has a certain effect.