Cloning and Prokaryotic Expression Analysis of Tyrosine Kinase Gene in Large Yellow Croaker

doi:10.13560/j.cnki.biotech.bull.1985.2016.11.021

Abstract

Abstract: To identify the structure and function of the lymphocyte-specific protein tyrosine kinase（LCK）gene（LcLCK）in large yellow croaker（Larimichthys crocea）,a full length cDNA of LcLCK was cloned from large yellow croaker by RT-PCR and RACE. The expressions of LcLCK in various tissues of adult male and female large yellow croaker as well as at different stages of the embryonic development were analyzed and examined via qRT-PCR. The recombinant prokaryotic vector was also constructed and transferred intoEscherichia coli for efficient expression. The results were as following：Its full-length cDNA sequence was 2 334 bp,with a 1 503 bp open reading frame encoding a protein of 501 amino acids. The predicted protein contained the typical domains of SH3,SH2 and TyrKc in the Src kinase family of non-receptor tyrosine. Two motifs of GCXCS and CXXC were found in the N-terminal region of LCK,while the two conserved Tyr sites in accordance with the Tyr³⁹⁴ and Tyr⁵⁰⁵ of LCK from human were present in C-terminal. Phylogenetic analysis showed that the deduced LCK clustered with Japanese pufferfish（Takifugu rubripes）. qRT-PCR revealed that the expressions of LcLCK were detected in all adult tissues examined,with higher expression in the main immune tissues such as spleen,head kidney,and gills of both sexes;and the expression level of LcLCK gene in these tissues from female was higher than that of the male. Early in the embryonic development,the high levels of LcLCK were observed during the multiple cells,blastula,and gastrula stages with the expression peak in blastula stage. The significant decrease of LcLCK expression was found at formation of yolk plug stage,and remained at the low expression level from formation of eye lens stage to pre-hatching stage,whereas an increase of gene expression was observed at alevin stage. The constructed prokaryotic vector pGEX-4T-2-LCK was expressed successfully in E. coli. In SDS-PAGE analysis,the new recombinant LcLCK protein band was about 84 kD that was in accordance with the expected. In conclusion,LcLCK is closely related to the cellular immune responses of adult male and female large yellow croaker as well as the mature of immune organs producing T-cell during embryonic development.

Key words: Larimichthys crocea, LCK gene, embryonic development, prokaryotic expression

ZHANG Zai-peng, LIN Peng, GUO Song-lin, WANG Yi-lei, ZHANG Zi-ping, FENG Jian-jun. Cloning and Prokaryotic Expression Analysis of Tyrosine Kinase Gene in Large Yellow Croaker[J]. Biotechnology Bulletin, 2016, 32(11): 180-187.

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