Variation Analysis Of WSSV And Construction Of Wsv089 And Wsv150yeast Two-hybrid Bait Vector

White spot syndrome has caused enormous losses to the shrimp farming industry worldwide since its outbreak.In order to understand the epidemic variation of WSSV in China,five high-variation regions ORF14/15,ORF23/24,ORF75,ORF94,ORF125,and wsv089,wsv150 were analyzed for variation,and yeast two-hybrid bait vectors of wsv089 and wsv150 were constructed in this study.The details are as follows:In this study,42 WSSV positive samples from regions of China were selected for PCR specific amplification of five variable regions of ORF14/15,ORF23/24,ORF75,ORF94 and ORF125 in 2017.Afterwards,the deletion variation and the change of single nucleotide polymorphism in the repeat unit were analyzed.The results showed that there were four deletion fragments in the amplification of ORF14/15,and the differences between the fragments were small.Only the 11945bp deletion fragment appeared in ORF23/24.In the ORF75 amplification,the total number of RUs was 3,4,and 9,and the 45bp RUs were polymorphic at the 12,27,and 80 positions.ORF94 only amplified a positive sample with a number of RUs of 6,and the single nucleotide polymorphism of each repeat unit at position 48.The number of RUs of ORF125 varies from 4,6,and 7.Base mutations occurred at positions of 20,27,50,53,and 61 in each ORF125 region.The results showed that a certain degree of deletion variation of WSSV was appeared in most parts of china,and some of the variable regions were showed the stability of the deletion.The number of repeating units in some variable regions and SNP were showed regional differences and instability.At present,there are relatively few studies on the genetic variation of wsv089 and wsv150 of different isolates of WSSV.In this study,42 WSSV positive samples from regions of China were selected for PCR specific amplification of wsv089 and wsv150 in 2017.Specific primers for the sequence were designed,and positive clones were selected for sequencing and analyzed for variation.The results showed that there were 10 positive samples in the PCR amplification of wsv089,which appeared in the samples of Xiamei Town of Fujian province,Zhanjiang(Guangdong Pro.),Dongying(Shandong Pro.)and Shanghai,with a detection rate of 23.8%.Compared with the standard sequence,the nucleotides of Xiamei Town in Fujian,Dongying in Shandong and Shanghai in Fujian were inserted into ATCT at position 72,resulting in the amino acid being mutated to leucine(L)at position 26 and inserted threonine(T),proline(P),serine(S)at position 27.In the samples of Zhanjiang area of Guangdong,only one nucleotide variation occurred,and CCCC was inserted at the 97th position.In the PCR amplification of wsv150,8 positive samples were detected,which was appeared in the Samples of Xiamei Town,Fujian Province,Zhanjiang,Guangdong,Dongying and Shanghai,with a detection rate of 19%.The nucleotide sequence was inserted into the base T at position 587,resulting in the substitution of 4 amino acids at positions196-199,the deletion of 106 amino acid fragments after the 200th amino acid,and the termination of amino acid translation.The yeast two-hybrid system has been widely used in the development of modern molecular biology in recent years due to its high sensitivity.It is a means of studying protein interactions in eukaryotic cells.In this chapter,the quality of the constructed cDNA library of intestinal tissue of Litopenaeu vannamei was analyzed.Furthermore,the bait vectors PGBKT7-089 and PGBKT7-150 was constructed,and the self-excitation and toxicity of the bait carrier were tested.The results showed that the cDNA library has a capacity of about 7.47×10~7 CFU/ml and the insert size is about500-2000bp,which meets the requirements of the library of Clontech's Yeastmaker~TMM yeast transformation system,and can be used in the next screening experiment.The colony PCR and sequence analysis indicated that the bait vectors PGBKT7-089 and PGBKT7-150 were successfully constructed and could not activate the expression of the reporter gene,They were not toxic to yeast cells and met the experimental requirements.It lays a foundation for screening host proteins interacting with wsv089 and wsv150.