Construction Of CDNA Library From The Skeletal Muscle Of Larimichthys Crocea And EST Analysis

Larimichthys crocea is important economic marine fish in China, facing a serious degradation of germplasm currently. By constructed a full length cDNA library, we can obtain a large number of muscle tissue functional information on gene expression in a short time,which were good enough for further screening and cloning of the specific expression genes in the skeletai muscle of Larimichthys crocea.A full length cDNA library from the skeletal muscle of Larimichthys crocea was constructed by using Creator Smart cDNA Library Construction Kit. The full length cDNA library was used for EST sequencing and bioinformation analysis.The main results were as follow:This library reached 1.6×106 cfu/ml in apacity; the percentage of recombinant efficiency was as high as 96%. PCR results showed that the average size of inserts was larger than 1000 bp, which was in full compliance with a cDNA library of standard.4006 clones were randomly selected and sequenced from 5'ends using primer M13. Of which, 3355 expressed sequence tags (ESTs) were high-quality, after removed low-quality sequence and vector sequence. The average length of the ESTs was 506bp and the average insertion length was 416bp. After searching NCBI and SWISSPROT protein database , 284 ESTs are expected to be novel genes because no sequence homology with any known sequences was found in GenBank batabases; 3071 ESTs with known function were identified by Blastx, on behalf of 317 homologous protein. Cytoskeleton-related genes of which had the highest abundance of ESTs, accounting for 27.68%, and energy production and conversion, signal transduction followed by the proportion of 22.82% and 20.68%, respectively. For single gene, the highest expression were myosin light chain, creatine kinase, myosin heavy chain,α-tropomyosin and cytochrome C oxidase.The full-length cDNA sequences of CD53 and interleukin-8 were successful cloned through designed primer, which were the first report in Larimichthys crocea.The aquired CD53 gene was 1,210bp in length, including a 113bp 5′-UTR, a 422bp 3′-UTR, and a 675bp coding sequence which encodes 224 amino acids. Theoretical molecular mass was 24.77 KDa and theoretical isoelectric point 5.07. The bioinformatics analysis showed that CD53 of Larimichthys crocea had four hydrophobic transmembrane spanning domains. Both its N-and C-termini were located in the interior of cell membrance, and two hydrophilic loops were located outside the membrance. Two N-glycosylated sites were located in the hydrophilic loop near the C-terminus. CD53 amino acid sequence was highly conserved, and the similarity among Gasterosteus aculeatus, Danio rerio, Oncorhynchus mykiss and Larimichthys crocea was over 70℅. Conventional RT-PCR was used to detect CD53 expression in adult of Larimichthys crocea. The results showed that CD53 was mainly expressed in intestinal, spleen, kidney, liver, muscle followed, except in adipose tissue, gill, eyes and brain.Il-8 gene was 2,582bp in length, including a 5′-UTR 106bp,a ExonⅠ52bp,a IntronⅠ133bp,a ExonⅡ133bp,a IntronⅡ149bp,a ExonⅢ87bp,a IntronⅢ682bp,a Exon IV13bp,a 3′-UTR 1191bp, and a 285bp coding sequence which encodes 94 amino acids. Theoretical molecular mass was 10.55 KDa and theoretical isoelectric point 9.64. The sequence contains four cysteine, of which the first two cysteines were separated by arginine, which formed CRC domain. The CXC domain was the characteristic of the chemotactic factor family. Conventional RT-PCR was used to detect IL-8 expression in adult of Larimichthys crocea..The results showed that IL-8 was mainly expressed in kidney, liver, intestinal, spleen, trace expression in heart, skeletal muscle and brain. IL-8 was not expressed in adipose tissue, gill and eyes.