大连海洋大学学报
大連海洋大學學報
대련해양대학학보
JOURNAL OF DALIAN FISHERIES UNIVERSITY
2014年
6期
550-555
,共6页
周贺%徐雯%李雅娟%秦艳杰%李霞%麻天宇%肖逸啸%马海艳
週賀%徐雯%李雅娟%秦豔傑%李霞%痳天宇%肖逸嘯%馬海豔
주하%서문%리아연%진염걸%리하%마천우%초일소%마해염
泥鳅%DNA甲基化%甲基化敏感扩增多态性 ( MSAP)%反应体系
泥鰍%DNA甲基化%甲基化敏感擴增多態性 ( MSAP)%反應體繫
니추%DNA갑기화%갑기화민감확증다태성 ( MSAP)%반응체계
Misgurnus anguillicaudatus%DNA methylation%methylation sensitive amplification polymorphism ( MSAP)%reaction system
为获得不同倍性泥鳅基因组DNA甲基化水平及模式,以泥鳅Misgurnus anguillicaudatus为研究对象,利用正交试验建立了甲基化敏感扩增多态性( MSAP)方法的反应体系,并利用该方法对二倍体泥鳅鳍基因组DNA进行了MSAP分析。结果表明：最佳双酶切反应体系为800 ng的泥鳅基因组DNA,用EcoR I、 Hpa II和Msp I各10 U,在37℃下反应8 h后即可酶切；最佳预扩增反应体系为模板4μL、预扩增引物0·8μL、0·2 mmol/L dNTPs、1·5 mmol/L Mg2+和 Taq 1 U；最佳选择性扩增反应体系为预扩增产物稀释20倍的模板2μL、引物1·5μL、0·375 mmol/L dNTPs、1·5 mmol/L Mg2+和 Taq 1 U；二倍体泥鳅全甲基化率分别为24·1%、20·8%、22·3%,半甲基化率分别41·4%、20·8%、33·3%,总甲基化率分别为65·5%、41·6%、55·6%。研究表明,该反应体系稳定、可靠、重复性好,为MSAP技术在多倍体泥鳅相关研究中的应用奠定了基础。
為穫得不同倍性泥鰍基因組DNA甲基化水平及模式,以泥鰍Misgurnus anguillicaudatus為研究對象,利用正交試驗建立瞭甲基化敏感擴增多態性( MSAP)方法的反應體繫,併利用該方法對二倍體泥鰍鰭基因組DNA進行瞭MSAP分析。結果錶明：最佳雙酶切反應體繫為800 ng的泥鰍基因組DNA,用EcoR I、 Hpa II和Msp I各10 U,在37℃下反應8 h後即可酶切；最佳預擴增反應體繫為模闆4μL、預擴增引物0·8μL、0·2 mmol/L dNTPs、1·5 mmol/L Mg2+和 Taq 1 U；最佳選擇性擴增反應體繫為預擴增產物稀釋20倍的模闆2μL、引物1·5μL、0·375 mmol/L dNTPs、1·5 mmol/L Mg2+和 Taq 1 U；二倍體泥鰍全甲基化率分彆為24·1%、20·8%、22·3%,半甲基化率分彆41·4%、20·8%、33·3%,總甲基化率分彆為65·5%、41·6%、55·6%。研究錶明,該反應體繫穩定、可靠、重複性好,為MSAP技術在多倍體泥鰍相關研究中的應用奠定瞭基礎。
위획득불동배성니추기인조DNA갑기화수평급모식,이니추Misgurnus anguillicaudatus위연구대상,이용정교시험건립료갑기화민감확증다태성( MSAP)방법적반응체계,병이용해방법대이배체니추기기인조DNA진행료MSAP분석。결과표명：최가쌍매절반응체계위800 ng적니추기인조DNA,용EcoR I、 Hpa II화Msp I각10 U,재37℃하반응8 h후즉가매절；최가예확증반응체계위모판4μL、예확증인물0·8μL、0·2 mmol/L dNTPs、1·5 mmol/L Mg2+화 Taq 1 U；최가선택성확증반응체계위예확증산물희석20배적모판2μL、인물1·5μL、0·375 mmol/L dNTPs、1·5 mmol/L Mg2+화 Taq 1 U；이배체니추전갑기화솔분별위24·1%、20·8%、22·3%,반갑기화솔분별41·4%、20·8%、33·3%,총갑기화솔분별위65·5%、41·6%、55·6%。연구표명,해반응체계은정、가고、중복성호,위MSAP기술재다배체니추상관연구중적응용전정료기출。
A reaction system of MSAP was established by orthogonal experiment in order to obtain genomic DNA methylation level and pattern of different ploidy of loach Misgurnus anguillicaudatus and used for MSAP analysis of diploid loach. The results showed that the best double digestion systems for MSAP including 10 U EcoR I, 10 U Hpa II, and 10 U Msp I were observed for 8 h at 37 ℃;the best pre-selective amplification mixture contained 0. 2 mmol/L dNTPs, 1. 5 mmol/L Mg2+, Taq 1 U and selective primers 0. 8 μL in total 20 μL;the best selective am-plification mixture contained 0. 375 mmol/L dNTPs, 1. 5 mmol/L Mg2+, Taq 1 U and selective primers 1. 5 μL in total 20 μL. The atlas showed that full methylation rate of three samples were 24. 1%, 20. 8% and 22. 3%, re-spectively;hemi-methylation rates were 41 . 4%, 20 . 8% and 33 . 3%, respectively;and total methylation rate was 65. 5%, 41. 6% and 55. 6%, respectively. The findings indicated that this MSAP reaction system was stable, reli-able and reproducible, providing the foundation for the MSAP technology with loach-related research.
