Identification and characterization of a biomineralization related gene PFMG1 highly expressed in the mantle of Pinctada fucata.
To elucidate the mechanism of nacre biomineralization, the mantle of Pinctada fucata (P. fucata) from the South China Sea was used. Using the mantle cDNA library and the ESTs we have cloned through suppression subtractive hybridization (SSH), ten novel genes including PFMG1 were obtained through nested PCR. Bioinformative results showed that PFMG1 had a high homology (40%) with Onchocerca volvulus calcium-binding protein CBP-1 and had two EF-hand calcium-binding domains from the 81st to the 93rd amino acid and from the 98th to the 133rd amino acid in the deduced amino acid sequence. The results of multitissue RT-PCR and in situ hybridization demonstrated the high expression of PFMG1 in the mantle of P. fucata and confirmed the SSH method. The results of GST-PFMG1 on CaCO3 crystallization showed significant effects on nucleation and precipitation of CaCO3. PFMG1 was cloned into the pcDNA.3.1/myc-HisA vector and was subsequently transfected into MC3T3-E1 cells. RT-PCR revealed upregulation of the marker genes related to cell growth, differentiation, and mineralization, and BMP-2, osterix, and osteopontin were upregulated as a result. This research work suggests that PFMG1 plays an important role in the nacre biomineralization, and the SSH method can pave the way for the bulk cloning and characterization of new genes involved in biomineralization in P. fucata and may accelerate research on the mechanism of pearl formation.
Liu
Hai-Luo
HL
Medical School, Tsinghua University, Beijing 100084, People's Republic of China.
Liu
Shang-Feng
SF
Ge
Ye-Jing
YJ
Liu
Jing
J
Wang
Xiao-Yan
XY
Xie
Li-Ping
LP
Zhang
Rong-Qing
RQ
Wang
Zhao
Z
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002113:Calcification, Physiologic; D020224:Expressed Sequence Tags; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D053495:Osteopontin / Q000096:biosynthesis; D049911:Pinctada / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis
D014157:Transcription Factors; D053495:Osteopontin