Molecular characterization and expression analysis of chitinase (Fcchi-3) from Chinese shrimp, Fenneropenaeus chinensis.
Complementary DNA (cDNA) and genomic DNA, including flanking regions of the chitinase gene (Fcchi-3) of Fenneropenaeus chinensis, were cloned and sequenced. Fcchi-3 was found to have 92.0 and 91.4% identity at the cDNA level to that of Litopenaeus vannamei and Marsupenaeus japonicus, respectively. The predicted amino acid sequence had an overall similarity with a comparable region of L. vannamei (96.8%) and M. japonicus (93.4%). Based on the cDNA sequence, the genomic structure of the gene was characterized. Sequence analysis revealed that the Fcchi-3 gene was composed of seven exons with 411, 252, 186, 132, 171, 117 and 135 bp and six introns with 232, 196, 121, 90, 159 and 157 bp. Analysis by RT-PCR revealed that Fcchi-3 was a hepatopancreas specific gene. Semi-quantitative RT-PCR analysis revealed that Fcchi-3 transcript was down-regulated significantly in response to the challenge of WSSV at 5 h post-injection and then came back to normal level at 37 h. A fusion protein containing Fcchi-3 was produced and the purified recombinant protein exhibited similar biological function. The result of identification through LC-ESI-MS showed that three peptide fragments (-MAADPVLR-, -ATIDPAYNVPELSK- and -AILAVGGWNEGSPK-) of the recombinant protein were identical to the corresponding sequence of L. vannamei chitinase-3. The recombinant Fcchi-3 could degrade the colloid chitin confirming that the recombinant protein is actually the chitinase.
Zhang
Jiquan
J
Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, People's Republic of China.
Sun
Yuying
Y
Li
Fuhua
F
Huang
Bingxin
B
Xiang
Jianhai
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002681:China; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons / Q000235:genetics; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D016678:Genome / Q000235:genetics; D043143:Hepatopancreas / Q000201:enzymology; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000201:enzymology* / Q000235:genetics* / Q000821:virology; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D046848:White spot syndrome virus 1 / Q000502:physiology
D018076:DNA, Complementary; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002688:Chitinases