Cloning, sequence, and function analyses of giant panda (Ailuropoda melanoleuca) CD9 gene.
CD9 is a member of the tetraspanin family proteins and has recently been shown to be essential for sperm-oocyte fusion in mice. The giant panda (Ailuropoda melanoleuca) CD9 (gpCD9) cDNA was amplified for the first time by RT-PCR from ovary total RNA and cloned, sequenced and analyzed. The result revealed that the open reading frame (ORF) of gpCD9 was 681 bp, which has the same length as that of mouse. Sequence analysis and structure prediction displayed that the amino acid sequence of gpCD9 is over 80% identity to those of mammals with the conserved structures, including the four transmembrane domains (TM) and certain characteristic residues. The results of sperm-egg fusion experiments demonstrated that giant panda CD9 large extracellular loop (LEL) significantly inhibited (P < 0.05) the mouse gamete fusion when the recombinant protein was added. However, when three amino acid residues TVT (173-175) of the gpCD9 were mutated to AAA, the large extracellular loop (LELM) of mutated protein was rarely inhibiting the gamete fusion of mice. Our results may be useful in improving an insight into understanding the potential mechanism of gamete fusion and genetic characteristics of giant panda.
Tang
Yang
Y
College of Life Science, Sichuan University, Sichuan Key Laboratory of Molecular Biology and Biotechnology, Chengdu, P. R. China.
Tan
Xue-Mei
XM
Yue
Chang-Wu
CW
Li
Cai-Xia
CX
Fan
Zhao-Xin
ZX
Zhang
Yi-Zheng
YZ
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology*; D003001:Cloning, Molecular; D005260:Female; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013084:Sperm-Ovum Interactions / Q000187:drug effects; D060245:Tetraspanin 29; D001503:Ursidae / Q000235:genetics*
D015703:Antigens, CD; C554870:Cd9 protein, mouse; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D060245:Tetraspanin 29