[Cloning and analysis of three genes encoding type II CHH family neuropeptides from Fennropenaeus chinensis].
On the basis of sequence similarity, the crustean hyperglycemic hormone (CHH) family peptides have been classified into two types of hormones: type I and type II. Molt-inhibiting hormone (MIH) is a neuropeptide member of type II CHH family. Molting in shrimp is controlled by MIH and ecdysone. By inhibiting the synthesis of ecdysone in the Y-organ, MIH indirectly suppresses the molting activity of shrimp. In this study, we reported the cloning and characterization of 3 gene fragments encoding type II CHH family neuropeptides of the shrimp Fennropenaeus chinensis. According to the complementary DNA sequence of the mult-inhibiting hormone of Fennropenaeus chinensis, 3 primers were designed and synthesized. MP1 and MP2 are sense primers, and MP3 is anti-sense primer. Polymerase chain reaction was performed using genomic DNA of Fennropenaeus chinensis as template. Three PCR products were obtained using primers MP1 and MP3. Their sizes are about 600 bp, 850 bp, 1050 bp, respectively. A 580 bp PCR product was obtained using primers MP2 and MP3. All the 4 PCR products were cloned into pMD18-T vector. The recombinant clones were sequenced using ABI 310 Genetic Analyzer. After sequencing, all the DNA sequences were searched in the GenBank by Blast program to find similar gene sequences. The searching results revealed 3 DNA fragment sequences were of high similarity with CHH family neuropeptide genes from various crustean species. The 3 DNA fragments were named as NP1, NP2, and NP3. Their sizes were 540 bp, 601 bp, and 826 bp, respectively. Using the mRNA sequences with the most similarity to the 3 sequence fragments as reference, the gene structure of the 3 DNA fragment sequences was analyzed. The exons of 3 sequence fragments were aligned with their similar sequences by Clustal W program. Both NP1 and NP2 consisted of 1 intron and 2 exons. NP3 consisted of 2 introns and 3 exons. Sequence analysis suggested that these 3 products belonged to sequence fragments of neuropeptide gene of type II crustacean hyperglycemic hormone family. The exons of NP1, NP2, and NP3 had highest similarity respectively with mRNA of Pem-SCP-C2 and Pem-SGP-C1 both from Penaeus monodon, and FenchMIH from Fennropenaeus chinensis. The identities were 91.5%, 92.8%, 88.9%, respectively. The results suggest NP3 is a fragment of molt-inhibiting hormone gene of Fennropenaeus chinensis NP1 and NP2 are two fragments of neuropeptide genes of type II crustacean hyperglycemic hormone family, which were found in Fennropenaeus chinensis for the first time.
Wang
Zai-Zhao
ZZ
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
Xiang
Jian-Hai
JH
D000595:Amino Acid Sequence; D060829:Arthropod Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D033364:Decapoda / Q000235:genetics*; D007447:Invertebrate Hormones / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009479:Neuropeptides / Q000235:genetics*; D010802:Phylogeny; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D060829:Arthropod Proteins; D007447:Invertebrate Hormones; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; C015947:hyperglycemic hormone, crustacean; D004247:DNA