CONSTRUCTION OF SUBSTRACTED cDNA LIBRARY OF LITOPENAEUS VANNAMEI INDUCED BY VIBRIO ALGINOLYTICUS AND ANALYSIS OF EXPRESSED SEQUENCE TAGS

Vibrio alginolyticus is one of the main causative agents resulting in serious infectious diseases of Litopenaeus vannamei and other animals.Suppression subtractive hybridization(SSH) is a new and highly effective method for analysing differentially expressed or tissue-specific cDNA probes and libraries.It is applicable to diagnosis of disease,development,tissue specificity,or other differential expressing.Based on the user manual of Clontech PCR-SelectTM cDNA subtraction kit,a suppression subtraction cDNA libraries of Litopenaeus vannamei were made by a PCR method.Experimental shrimp were injected with 5×107 CFU live Vibrio alginolyticus and the control shrimp were injected with steriled normal saline.The mRNA from hemocytes of normal shrimp and vibrio-challenging shrimp must be isolated firstly.After mRNA was reverse transcribed into double strand cDNA,the two kinds of ds cDNA were denominated with Driver cDNA(normal shrimp) and Tester cDNA(vibrio challenging shrimp) separately.Then they were digested with Rsa I at the same time but only Tester ds cDNA were diluted and ligated to adaptor l and adaptor 2R in separate ligation reactions in the same total volume.Then an excess of driver cDNA was added to each tester cDNA for the first round of hybridization to enrich for differentially expressed sequences.Secondly,the two samples from the first hybridization were mixed together and freshly denatured driver DNA was added to each tester cDNA for differentially expressed sequences.Only the remaining normalized and subtracted ss tester cDNA were able to reassociate and form newly hybrids with different adaptors at their 5'-ends in the second hybridization.The new hybrids were preferentially amplified by PCR with a pair of primers,nested PCR primer 1 and nested PCR primer 2R.Litopenaeus vannamei β-actin gene was used as internal control to estimate the efficiency of subtractive successfully constructed.In this library,β-actin was subtracted significantly above 10 cycles,suggesting that the subtractive cDNA library was successfully constructed.The substracted products were cloned into the pMD-T 18 Vector.Then the recombinated plasmid was transformed into DH5α.PCR analysis showed that the inserts were 488bp equally in length.The subtracted cDNA library including 2000 clones and picked 600 clones to sequence randomly.The obtained 560 ESTs were assembled to 239 Unigenes(164 contigs and 75 singletons) with DNAMAN 5.2.2 software.Compared with sequences in unigene database of GeneBank with BLASTx and BLASTn algorithm,159 ESTs of them had comparatively clear results and the percent of them in acquired ESTs was 66.9%.The sequences of these ESTs were subjected to GO annotation.According to their physioligical function,they could be subdivided into 7 categories,36% were metabolism genes;15% were immune-related genes;8% were others genes;3% were regulation and signal transaction factors;2% were apoptotic-related proteins and antioxidant enzyme;1% were ribosomal proteins.The result showed that Litopenaeus vannamei,induced by Vibrio alginolyticus,could express serials of special genes.Analysis of the libraries indicates the PCR based suppression subtraction cDNA libraries is feasible used to discover the immune gene in shrimp.