Cloning of Stage2specific Gene Fragments in Rabbit Embryos

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1 Chinese Journal of Zoology 2009,44 (4) : ( ; ; ) :, mrna ( Oryctolagus cuniculus domestica) 42, 5 NCBI EMBL, EMBL,, 8 16, : ;mrna ;; :Q132 :A : (2009) Cloning of Stage2specific Gene Fragments in Rabbit Embryos QI Bing CHANG Zheng2Yao LI Xiao2Liang LI Yong2Fang CHEN Qing2Xuan LI Wen2Yong 3 ( Department of Biological Sciences, Taishan Medical University, Taian, Shandong ; Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing ; Department of Biology, Fuyang Normal College, Fuyang, Anhui , China) Abstract: Proper expression of stage2specific genes is very important for early embryo development. The mrna differential display was used to study the stage2specific genes in rabbit preimplantation embryos. A total of 42 positive differential fragments were obtained,of which 5 fragments had no homolog in the NCBI and EMBL. These fragments were submitted to the EMBL and accession numbers were given. All these 5 genes were specifically expressed in morula stage. Maternal2to2zygotic transition of rabbit starts from 8-16 cell stage,and therefore,these genes should be embryo2specific. This research would lay a foundation for further investigating the transcriptional modulation of rabbit preimplantation embryo development. Key words : Rabbit ( Oryctolagus cuniculus domestica) ; mrna differential display ; Morula stage embryo ; Zygotic gene, (transcription2requiring complex, TRC) ( Mus musculus domesticus) 22,4 %, (No ) ; 3,E2mail com. cn ;,,,; : ; E2mail : com. cn : , :

2 42 Chinese Journal of Zoology 44, 4 2 [1 ] Rnf35 ( RING2 finger), [2 ], [3 ],,, [4 ],, mrna mrnas,, 22 [5 ], ( Oryctolagus cuniculus domestica) 8 16 [6 ],, mrna, 1992 Liang mrna,, [7 ] 1994 DDRT2PCR [8 ],, [9 15 ] mrna,, , DH5, p GEM2T PCR, Promega ; Taq DNA Promega ;Superscript GIBCO BRL, RNeasy Mini Kit for the Isolation of RNA Qigen ; SuperScrip TM First2Stand Synthesis System for RT2PCR GIBCO FSH ( ),hcg( ) ; ( Sigma ), Glycogen ( Roche ) ;Primer RNase ( Eppendorf),DNase GibcoΠBRL ), RNA Guard ( Amersham Pharmacia Biotech),Oligo (dt)218 (MBI Fermentas) [ 2 32 P] datp (3 000 CiΠmmol) 3 HT(15) G 5 HAP1 (5 2AAGCTTGATTG2 CC23 ) , 6 FSH ( ), 5 IU, 12 h, FSH 12 h, 100 IU hcg( ) hcg 13 h,, PBS, M (300 IUΠml), PBS 3 hcg, 13 h 12 ( ) 24 h 22,40 h 42,54 h 8 16,82 h, 94 h mrna (DDRT2PCR) RNA (5 ) PBS, rπ min 30 min,, ; 600 l RLT buffer, 2 min,,,; 600 l RW1, ;, 500 l RPE,; ; 30 l DNase2RNase, 5 min RNA DNA RNA DNase,37 30 min, Π2

3 4 : 43,3 molπl - 20,70 % 2, 20 l DNase2RNase l RNA, 2 l 10 mmolπl Oligo (dt)218,70 5 min, 2 min 5 first buffer 10 l, RNA Guard 1 l,primer RNase 1 l,011 molπl DTT 2 l,10 mmolπl dntp 2 l, Superscript 1 l, 50 l, h 70 5 min PCR 5 l PCR, 20 l, 10 PCR 2 l,2 l 25 mmolπl MgCl 2,2 l 25 molπl HT (15) G,2 l 25 molπl HAP1,015 l [ 2 32 P ] datp,1 l 1 molπl dntps, 015 l Taq DNA,RNase2DNase 20 l PCR : min,40 5 min,72 5 min ;94 30 s,40 2 min, min, 39 ;, 72 5 minpcr PCR 8 molπl 6 % ( 3316 g,40 % 8 ml,10 TBE 8 ml, 32 ml,80 ml, 350 l 10 % APS 35 l TEMED) 4 5 h, 40 W, - 20 X, 2 3 d X,,, 100 l, 10 min, min, g 2 min,, 8 l (3 molπl) 300 l 215 l (20 mgπml), - 20,, 10 l DEPC 50 l ( , [ 2 32 P] datp, HT(15) G HAP1), PCR : min ;94 1 min,40 90 s,72 90 s,20,72 10 min ;94 10 min ;94 1 min, 115 % PCR PCR,p GEM2T, 10 l (1 T4 DNA, p GEM2T 50 ng,pcr 3 l,t4 DNA 30 U),,4 (CaCl 2 ) JM109, 200 l JM ml EP, 3 l,, 30 min,42 90 s, 2 min, 800 l LB (),37 1 h, JM l 4 l IPTG (200 mgπml) 16 l X2gal (50 mgπml), (100 gπml) LB, 37, 3 ml LB,37, PCR,, NCBI, EMBL mrna, 1 1 2,, DNA :, , %, 213 GenBank s,72 90 s,20 ; min 60

4 44 Chinese Journal of Zoology 44 5 EST 3 1 Table 1 Accession number of new fragments Clone number (bp) Size of fragments Accession number S AJ S AJ S AJ S AJ S AJ mrna Fig. 1 mrna differential display of rabbit preimplantation embryos 11 ( ) ; 21 ( ) ; 31M ; ; ; ; ; 81 ; Negative control (transcription template is water) ; 21 Negative control (without reverse transcriptase) ; 31 M oocyte ; 41 12cell embryo ; 51 22cell embryo ; 61 42cell embryo ; cell embryo ; 81 Morula ; 91 Blastocyst. 2 Fig. 2 Agarose electrophoresis of differential fragments M. DL2000 DNA ; M. DL 2000 DNA marker ;Others are sub2cloned fragments. HT(15) G HAP1 42 GenBank, BLAST nr EST 5 nr EST, GenBank ( 1) 5,, 3, 3 :, ; ;,, [16 ] mrna, mrna, mrna 22, 82, mrna,, 42 5 nr EMBL, 5,,, mrna,3,,

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