Nucleotide Sequence of Cloned cd NA for alpha Subunit of Goat Follicle Stimulating Hormone

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 18 (6) : cdna, 3, (, ) RNA, cdna cdna PCR, 380 bp cdna pmd2182t2verctor 3,,, 96 %, 95 %,, 74 %, 95 %, 94 %,, 85 %, FSH,,cDNA, Q78 Nucleotide Sequence of Cloned cd NA for alpha Subunit of Goat Follicle Stimulating Hormone ZHANGLi, LI Qing2zhang 3, GUAN Hong2bin ( Department of Veterinary Medicine, Northeast Agricultural University, Harbin , China) Abstract Total RNA was prepared from pituitary gland of new butchered female goat cdna was synthesized by RT2PCR and was used as a model in PCR amplification for 2subunit of goat follicle stimulating hormone The PCR product was 380 bp as determined by 1 2 % agarose gel electrophoresis with the same length as FSH 2subunit After cloning it to pmd2182t2vector and selecting 3 positive recombinants at random the DNA se2 quences were then analyzed Comparison of their amino acid sequences with those of other mammalian species showed that goat had the highest homology of 96 % with sheep and buffalo with only 5 amino acids difference ; and high homology of 95 % with bovine ;and lower homology of 74 % with human It had the highest homology of 95 % with sheep in nucleotide sequence,and high homology of 94 % with bovine and buffalo,and lower ho2 mology of 85 % with horse and rat It was found that the nucleotide sequence of the FSH 2subunit in these mammalian species were highly conservative Goat FSH 2subunit gene was reported Key words goat follicle stimulating hormone, 2subunit,cDNA,nucleotide sequence analysis (follicle stimulating hormone,fsh),,, 30 kd, 2,2, N2 [1 ] 92, 115 FSH, : ; : Tel : ,E2mail com [2,FSH ] (LH) ( TSH) (CG),,,1978, Received :January 15,2002 ;Accepted :March 27, Corresponding author Tel : E2mail com

2 [3,4 ], JM109,,, PCR Hind EcoR,,,, ( ) [5 ] FSH, 2 ; FSH, 211 RNA G, Trizol Kit RNA camp,, 28 S 18 S rrna [6 ] FSH [7 ], RNA Fig 1 FSH [8 ] [9 ; ] [10 ] [11 ] [12 ] [13 ] [14 ], FSH FSH cdna, FSH Takara RNA PCR Kit (AMV) Ver 2 1, Trizol Re2 agent Kit, Taq TM,, PMD2182T2Vector ( ) JM109, RNA,, 112 RNA RNA Trizol Reagent Kit Trizol, EP RNA DEPC, cdna oligo (dt) 15, Poly(A) + RNA cdna Takara RNA PCR Kit (AMV) Ver PCR cdna 380 bp DNA, FSH, Fig 2, ( ) :5 2C C CAA GC T TAA GC C A T G GA T TA C TA CA GA A A23 ; 2 :5 2C C G G DNA Fig 1 Agarose gel electrophoresis of goat total RNA 1,2 : Goat total RNA 212 PCR cdna PCR FSH cdna, Fig 2 :94, 5 min ;94, 30 s,55,30 s,72,45 s,72,10 min, PCR PCR pmd2182t2verctor, JM109, 6 PCR A A T T C T TA A GA T T T GT GT GA TA A TA A CA2, PCR Fig 3 3 RT2cDNA, FSH 380 bp ;, cdna dsdna 1 2 % Hind EcoR,, Fig PCR FSH DNA RT2PCR FSH2 cdna, Fig 5, pmd2182t2vector

3 6 : cdna 695 Fig 2 PCR amplification of 2subunit gene 1,2,3,5,6 :Products of PCR ; 4 : DL2000 marker Fig 3 Identification of recombinant FSH 2subunit gene by PCR 1 6 : 2Subunit gene ; 7 :DL2000 marker 214 FSH Fig 6 Table 1 Table 1, FSH, 96 %, 5, 95 % ;, 74 % FSH,, 95 %, 94 %, 85 % Fig 4 Double enzyme digestion of recombinant plasmid 1 :DL2000 marker 2 :Recombinant plasmid digested by Hind + EcoR Fig 5 The gene sequence of goat FSH 2subunit (5 3 )

4 Fig 6 Amino acid sequence comparison among goat and other animals Table 1 Homology of nucleotide and amino acid sequence comparison among goat and other animals goatπsheep goatπbuffalo goatπbovine goatπpig goatπdog goatπtiger goatπrat goatπdonkey goatπhorse goatπhuman Amino acid sequence 96 % 96 % 95 % 93 % 91 % 91 % 88 % 82 % 78 % 74 % Nucleotide sequence 95 % 94 % 94 % 92 % 90 % 89 % 85 % 87 % 85 % ( References) 1 ( Wang Jian2chen ( Gou De2ming,Li Wen2xin, Huang Jian, Xia Zong2ping,Li Zhi, Jiang Da2he The screening and sequencing of human chorionic gonado2 tropin and 2subunit cdna Chin J Biochem Mol Biol),1999,15 (6) : Hirai T, Takikawa H, Kato Y Molecular cloning of cdna for precur2 sors of porcine pituitary glycoprotein hormone common 2subunit and of thyroid stimulating hormone 2subunit Mol Cell Endocrinol,1989,63 : , (Li Feng2e, Xiong Yuan2zhu Survey on FSH gene study Animal Seience Broad :Pigs and Poultry),2001, (1) : ,,,, ed Domestic Animal Reproduction Endocrinology Beijing : Agriculture Press) 1993 : , ( Fan Jun,Deng mrna (Ding Yun2min, Ye Shi2jun,Chen Yong2mei,Zheng Wen2li, He Xing2hua The postnatal changes of follicle2stimulating hormone re2 Xiao2hui The new progressing on follicle2stimulating hormone Repro2 ceptor mrna level in rat testis Acta Acad Med Sci),1996,18 (2) :99 duction & Contraception),1998,18(2) : ,,, 7,,,,, cdna cdna ( Xi Fen2gong, Yu Hong, Chen Xin2guo,Shi Yu2hu,Chen Qing2xuan,Zhang Zhi2hong Nucleotide sequence of cloned cdna for 2subunit of bovine follicle stimulating hormone Prog Biochem Biophys),1998,25 (2) : ,,,,,, B (Ai Xiu2lian, Chen Xin2guo,Ji Qing,Long Tao, Yu Hong,Xi Fen2gong,Huang Ping, Shi Yu2hu The study of using biotechnigue to produce bovine FSH Sci2 ence in China, Series B),1995,25 (6) :

5 6 : cdna Fiddes J C, Goodman H W Isolation,cloning and sequence analysis of the 2subunit of human chorionic gonadotropin Nature (London),1979, 281 (4) : Erwin C R,Croyle ML,Donelson J E, Maurer R A Nucleotide sequence of cloned complementary deoxyribonucleic acid for the 2subunit of bo2 vine pituitary glycoprotein hormones Biochemistry, 1983,22(20) : Chopineau M, Stewart F Cloning, sequence analysis of the cdna for the common alpha2subunit of the donkey pituitary glycoprotein hor2 mones J Mol Endocrinol,1996,16(1) : Toshiaki Hirai,Hiroo Takikawa,Yukio Kato Molecular cloning of cdna of porcine pituitary glycoprotein hormone common 2subunit and of thy2 roid stimulating hormone 2subunit Mol Cell Endocrinol,1989,63 : Pinki Garg,Jain S K Molecular cloning of a cdna for the beta subunit of sheep follicle stimulating hormone Med Sci Res,1992,20 : Godine J E,Chin W W,Habener J F 2Subunit of rat pituitary glycopro2 tein hormones J Biol Chem,1982,257 (14) : ,, 2003, DNA 50 30,, 30 1 :, :, 2 : ; : : 3 : : : 5 : (1) 30 ( 10 5 ), ( ) (2) DNA 50 ; ; ; 6 : 7 :, 8 :,,, E2mail ac cn / : : : 917,, ( ),