48 (6) : , ( Follicle2Stimulating Hormone, FSH) ( Fauser, 1998) (L uteinizing Hormone, L H)

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1 48 (6) :783789, 2002 A cta Zoologica S inica 3 33 (, ) (, ) ( Panthera tigris altaica) RNA, RT2PCR ( FSH) (L H),, FSH L H 363 bp 390 bp 429 bp, ,, (6417 %9616 %), (86 %9616 %), L H cdna (Leu) FSH L H ( Follicle2Stimulating Hormone, FSH) (L uteinizing Hormone, L H),,,,,,, ( Pierce et al., 1981) (, 1998), ( Gonadotropin Releasing Hormone, GnRH),, FSH L H,, ( Gharib et al., ( FSH) (L H) 1990 ; Margaret et al., 1996),, FSH, L H, FSH,, FSH L H ;, L H FSH ( Gharib et al., 1990), FSH L H, FSH 111 L H, 11111,,,, FSH L H, J M109 FSH L H (Li et EcoR Pst al., 1998),, FSH, PCR, RNA L H GIBcoBRL TRIzol g Reagent, , ( ) 33 E2mail, ( Fauser, 1998),, 25, :,,, 1

2 RNA 1 % 2 RT2PCR Fig11 1 % formaldehyde agarose gel electrophoresis analysis of total RNA M : DL 2000 (DL 2000 Marker) A : RNA ( Total RNA) Promega Reverse Transcription Fig12 Electrophoresis analysis of RT2PCR amplif ied products M : DL 2000 (DL 2000 Marker) A : (subunit product) B : FSH ( FSHsubunit product) C : L H (L Hsubunit product) SystemPCR GTG 3, Pfu DNA, TFB 1 : 5 CAA GTG CCC A GG A TG AA G 3 puc18 TFB 2 : 5 CTC CAC TGC TCT TTA TTC TLB 1 : 5GGA TGG A GA TGC TCC A GG GAC RNA 200 mg TG 3,,, TLB 2 : 5 GGG GCA TCC TTA GA G GAA CGG G TRIzol g Reagent RNA 3 RNA DEPC, RT2PCR RNA, 1 % Oligo dt, Promega RNA - 70 cdna PCR ( Fiddes et min, 95 5 min, 5 5 min al., 1979 ; Talmadge et al., 1984 ; Wat kins et er et al., 1987) ( Chin et al., 1981 ; Ku2 CTA 3 TGA 2 : 5 CAC GGC AAA CTA T TA GGA T T T, TGA1 al., 1987) ( Erwin et al., 1983 ; Maurer et al., 1985 ; Esch et al., 1986) (Bello et TGA2, TFB1 TFB2, TLB1 TLB2 PCR 50 l, 115 al., 1989 ; Mountford et al., 1989 ; dπangelo2 mmol/ L MgCl 2, 200 mol/ L dn TP, Bermard et al., 1990) ( Hirai et al., 1989 ; 013mol/ L 5 U Pfu PCR Ezashi et al., 1990 ; Li et al., 2000 ) : 94 3 min ; s, 50 (Chin et al., 1983 ; Burnside et al., 1988 ; Maur2 40 s, s, 30 ; 72 5 minrt2pcr 116 % mar et al., 1994 ; Kumar et al., 1995) PCR Gene Amp PCR FSH L H cd2 System 9700 ( PE ) NA, PCR PCR, :, 3 1 TGA 1 : 5 A GG ACG AA G A GC CA T GGA TTA S m a puc18 22 (J M109)

3 6 : 785, 50 g/ ml 200 mg/ 3 DNA (3), ml IPTG20 mg/ ml X2Gal LB, FSH L H,, PCR 387 bp (3 A) 414 bp (3 B) 440 bp (3 C), WD2 DNA, AB I WDNAsisGENDOC, DNA GenBank, 10 FSH L H (Cys) 2 FSH RNA RNA A260/ A , , 18 S 28 S 12 ( Cys) 1 RNA (1), ORF GenBank ( RNA, : AF408393, AF540937, AF540935) 212 RT2PCR PCR, ( TGA1 TGA2, TFB1 TFB2 TLB1 TLB2) RT2PCR, ( FSH L H DNA ) 116 %, (86 %9616 %) (2), PCR, 390 bp ( ) 420 bp ( FSH ) 440 bp (L H ) PCR - - puc18, J M109, FSH,,, L H, PCR, FSH L H, 213 NAsis, 120, 24 96, 129, 19, 12 (Cys) 2 L H 429 bp, (1), FSH L H (6417 %9616 %), FSH L H 3 (Li et puc18, al., 1998), 1 FSHL H ( %) Table 1 Comparison of homology of Panthera tigris altaica subunit, FSHand L Hwith other reported mammals at the nt level and the aa level ( %) Gene Level for comparision Human Bovine Sheep Pig Rat Mouse (nt) (aa) FSH (nt) (aa) L H (nt) (aa)

4 FSH L HcDNA Fig13 cdna sequence and deduced amino acid sequence ofandsubuint of FSH and L H from Panthera Tigris altaica A. (subunit) B. FSH ( FSHsubunit) C. L H (L Hsubunit) ( The primer se2 quences are underlined) ( The signal peptides are double underlined) N2 ( The N2glyco2 sylation sites are boxed by open rectangles) ( The asterisks represent stop codens) (6417 %9616 %), (Cys) N2, (4),, Cys, Asn, ( Gharib et al., 1990), FSH L H, (Brown et al., 1996 ; Howard et al., 1997),, FSH L H FSHL H, (4), [ Phillips et al., 1982 ] 39, V, I, (4 A) ; FSH 20, 24, 68 96, M, A, Q, Q, N,,,, I, H, R (4 B) ; L H 21,

5 6 : FSH L H Fig14 Alignment of andsubunit putative aa of FSH and L H of Panthera tigris altaica with those of other species A. (subunit) B. FSH ( FSHsubunit) C. L H (L Hsubunit) tiger ( tigerrepresents Panthera tigris altaica) (Dots indicate cysteine residues) N2 ( The arrows indicate potential N2 linked glycosylation sites), T, V, D, A, I, L (4 C), - 12, Leu (L), L H,,,

6 ( References) Bello, P. A., J. R. McNeilly, M. R. Brandon and T. E. Adams 1989 Cloning and DNA sequence analysis of the cdna for the common alpha2subunit of the ovine pituitary glycoprotein hormones. N ucleic Acids Res. 17 : Brown, J. L., D. E. Wildt, N. Wielebnowski, K. L. Goodrowe, L. H. Graham, S. Wells and J. G. Howard 1996 Reproductive ac2 tivity in captive female cheetahs ( Aci nonyx j ubat us) assessed by faecal steroids. J. Reprod Fertil. 106 : Burnside, J., P. R. Buchl and W. W. Chin 1988 Isolation and characterization of the gene encoding thesubunit of the rat pituitary glycopro2 tein houmones. Gene 70 : Chin, W. W, J. E. Godine, D. R. Klein, A. S. Chang, L. K. Tan and J. F. Habener 1983 Nucleotide sequence of the cdna encoding the precursor of the beta subunit of rat lutropin. Proc. N atl. Acad. Sci. USA 80 : Chin, W. W., H. M. Kronenberg, P. C. Dee, F. Maloof and J. F. Habener 1981 Nucleotide sequence of the mrna encoding the pre2 2 subunit of mouse thyrotropin. Proc. N atl. Acad. Sci. USA 78 : dπangelo2bermard, G., M. Moumni, M. J utisz and R. unit of ovine luteinizing hormone. N uceic Acids Res. 18 : Counis 1990 Cloning and sequence anlysis of the cdna for the precursor of the beta sub2 Erwin, C. R., M. L. Croyle, J. E. Donelson and R. A. Maurer 1983 Nucleotide sequence of cloned complementary deoxyribonucleic acid forsubunit of bovine pituity glycoprotein hormone. Biochemist ry 22 : Esch, F. S, A. J. Mason, K. Cooksey, M. Mecado and S. Shimasaki 1986 Cloning and DNA sequence analysis of the cdna for the precur2 sor of the beta chain of bovine follicle stimulating hormone. Proc. N atl. Acad. Sci. USA 83 : Ezashi, T., T. Hirai, T. Kato, K. Wakabayshi and Y. Kato 1990 The gene for the beta subunit of porcine L H : clusters of GC boxes and CACCC elements. J. Mol. Endocri nol. 5 : Fiddes, J. C and H. M. Goodman 1979 Isolation, cloning and sequence analysis of cdna for thesubunit of human chorionic gonadotropin. N at ure 281 : Gharib, S. D., M. E. Wierman, M. A. Shupnik and W. W. Chin 1990 Molecular biology of the pituitary gonadotropins. Endocri ne Re2 views 11 : Howard, J. G., T. L. Roth, A. P. Byers, W. F. Swanson and D. E. Wildt 1997 Sensitivity to exogenous gonadotropins for ovulation induction and laparoscopic artificial insemination in the cheetah and clouded leopard. Bio. Reprod. 56 : Hirai, T., H. T. Akikawa and Y. Kato 1989 Molecular cloning of cdnas for procusors of porcine pituitary glycoprotein hormone common alpha2subunit and of thyroid stimulating hormone beta subunit. Mol. Cell. Endocri nol. 63 : Kumar, T. R., M. Kelly, M. Mortrud, M. J. Low and M. M. Matzuk 1994 Cloning of the mouse gonadotropin 2subunit2encoding genes,. structure of the follicle2stimulating hormone 2subunit2encoding gene. Gene 166 : Kumar, T. R and M. M. Matzuk 1995 Cloning of the mouse gonadotropin beta2subunit2encoding genes, : structure of the luteinizing hormone beta2subunit2encoding genes. Gene 166 : Li, M. D., G. A. Rohrer, T. H. Wise and J. J. Ford 2000 Identification and characterization of a new allele for the beta subunit of follicle2 stimulating hormone in Chinese pig breeds. A ni m Genet. 31 (1) : Li, M. D and J. J. Ford 1998 A comprehensive evolutionary analysis based on nucleotide and amino acid sequences of theand subunits of gly2 coprotein hormone gene family. Journal of Endocri nalogy 156 : Ma, Y. Q and W. Yan 1998 Advances in the tiger protection. Chi nese Wil dlif e 19 (1) : 37. [, (1) : 37. ] Margaret, A. S Gonadotropin gene modulation by steroids and gonadotropin2releasing hormone. Biology of Reproduction 54 : Maurer, R. A Analysis of several bovine lutropin beta subunit cdnas reveals heterogeneity in nucleotide sequence. J. Biol. Chem. 260 (8) : Maurer, R. A Molecular cloning and nucleotide sequence analysis of complementary deoxyribonucleic acid for the beta2subunit of rat follicle2 stimulating hormone. Mol. Endocri nol. 1 : Mountford, P. S., P. A. Bello, M. R. Brandon and T. E. Adams 1989 Cloning and DNA sequence analysis of the cdna for the precursor of ovine follicle stimulating hormone 2subunit. N ucleic Acids Research 17 : Phillips, L. G., L. G. Simmons, M. Bush, J. G. Howard and D. E. Wildt 1982 Gonadotropin regimen for inducing ovarian activity in captive wild felids. J. A m. Vet. Med. Assoc. 181 (11) : Pierce, J. G and T. F. Parsons 1981 Glycoprotein hormone : structure and function. A nnu. Rev. Biochem. 50 : Talmadge, K., N. C. Vamvakopoulos and J. C. Fiddes 1984 Evolution of the genes for thesubunits of human chorionic gonadotropin and luteinizing hormone. N at ure 307 : Watkins, P. C., R. Eddy, A. K. Beck, V. Vellucci, B. Leverone, R. E. Tanzi, J. F. Gusella and T. B. Shows 1987 DNA sequence and regional assignment of the human follicle2stimulating hormone beta2subunit gene to the short arm of human chromosome 11. DNA 6 (3) :

7 6 : 789 ( Abstract) CLONING AND ANALYSIS OF THE HOMOLOGY OF THE FOLL ICL E2STIM ULATING HORMONE AND L UTEINIZING HORMONE GENES IN SIBERICAN TIGER ( PAN TH ERA TI GRIS AL TAICA) 3 L IAO Ming2J uan ZHU Mu2Yuan 3 3 YE Dan ZHAN G Zhi2He ZHAN G An2J u SHEN Fu2J un ( College of L if e Sciences, Zhejiang U niversity, Hangz hou , Chi na) ( Chengdu Research Base of Giant Panda B reedi ng, Chengdu , Chi na) The pit uitary gonadot ropin follicle2stimulating hormone ( FSH) and luteinizing hormone (L H) play impor2 tant roles in regulating gonad f unction and are essential for normal reproductive f unction in mammals. In t his pa2 per, by comparing the FSH/ L H common, FSHand L HcDNA sequences reported from humans, cattle, sheep, pigs, rats and mice, three pairs of primers were designed and synthesized according to the conserved re2 gion. Using these primers, the genes encoding, FSHand L Hin Panthera tigris altaica were amplified by reverse t ranscript polymerase chain reaction ( R T2PCR) f rom pit uitary total RNA and were cloned, sequenced and submitted to GenBank, wit h accessions Nos. AF408393, AF and AF respectively. It was re2 vealed that the open reading region (ORF) of subunitfrom Panthera tigris altaica was 369 bp encoding a pre2 cursor containing a 24 amino acid signal peptide and a 96 amino acid mature peptide. Similarly, the ORF of FSH was 390 bp encoding a 19 amino acid signal peptide and 110 amino acid mature protein. The ORF of the L H subunit was 429 bp encoding a 141 amino acid protein which had an additional Leu residue in the 21 amino acid signal peptide sequence compared to other species. The mature protein of the L Hsubunit contained 121 amino acids just like those of other species. The predicted primary structure of the three subunits were similar to those of other mammals with conserved cysteines (10 for thesubunit and 12 for thesubunit) and putative N2glyco2 sylation sites (2 for theand FSHsubunits, 1 for the L Hsubunit). When nucleotide sequences and deduced amino acids were compared wit h homologous sequences f rom available mammals including humans, cattle, sheep, pigs, rats and mice, they displayed a fairly high (6417 %9616 %) degree of conservation. Among all species compared, pigs exhibited the highest degree of amino acid similarity of 9616 %, 9115 % and 8616 % with Pant hera ti gris altaica. Sequence alignment indicative of some distinct amino acid diversity, which could be re2 lated to t he species2specific characteristics of gonadot ropins, was also found in t he Pant hera ti gris altaica se2 quence, such as position 39 of thesubunit, 20, 24, 68 and 96 of the FSHsubunit and 27 and 119 of the L H subunit. Our results not only provide an insight into the characteristics of FSH and L H in Panthera tigris al2 taica that can make a practical contribution to its conservation, but also lay the foundation for further research towards producing recombinant FSH and L H which could increase t he efficiency of artificial breeding technolo2 gy. Key words Panthera tigris altaica, Cloning, Assisted reproduction, FSH and L H 3 This work was supported by the Chengdu Research Fund of Giant Panda Breeading ( ) 33 Corresponding author.

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