分子植物育种
分子植物育種
분자식물육충
MOLECULAR PLANT BREEDING
2013年
5期
611-616
,共6页
谢一青%黄勇%卓仁英%李志真%姚小华
謝一青%黃勇%卓仁英%李誌真%姚小華
사일청%황용%탁인영%리지진%요소화
油茶%cDNA-AFLP%体系优化%引物筛选
油茶%cDNA-AFLP%體繫優化%引物篩選
유다%cDNA-AFLP%체계우화%인물사선
Camellia oleifera%cDNA-AFLPR%System optimization%Primer selection
以小果油茶和普通油茶的嫩叶及种仁为材料,对影响其cDNA-AFLP反应体系的关键因子进行优化分析,筛选出适合油茶的cDNA-AFLP反应体系。结果表明:分别用EASYspin Plus植物RNA快速提取试剂盒和Trizol法能成功获得高质量的油茶嫩叶和种仁总RNA;从总RNA中分离的mRNA经合成双链cDNA后,用限制性内切酶EcoRⅠ和MseⅠ在37℃下5 h内酶切完全,经5 U T4连接酶16℃连接的产物稀释10倍后可直接用于预扩增;在20μL选择性扩增体系中,以预扩增产物稀释30倍为模版、dNTP (10 mmol/L)1.5μL、引物(10μmol/L)1.0μL、Taq酶用量1.25 U时的扩增效果较好。利用优化体系成功筛选出61对可以获得带型丰富且重复性好的引物组合。差异片段回收及PCR检测的结果表明,优化后的cDNA-AFLP分析体系适用于油茶相关差异基因的表达研究。
以小果油茶和普通油茶的嫩葉及種仁為材料,對影響其cDNA-AFLP反應體繫的關鍵因子進行優化分析,篩選齣適閤油茶的cDNA-AFLP反應體繫。結果錶明:分彆用EASYspin Plus植物RNA快速提取試劑盒和Trizol法能成功穫得高質量的油茶嫩葉和種仁總RNA;從總RNA中分離的mRNA經閤成雙鏈cDNA後,用限製性內切酶EcoRⅠ和MseⅠ在37℃下5 h內酶切完全,經5 U T4連接酶16℃連接的產物稀釋10倍後可直接用于預擴增;在20μL選擇性擴增體繫中,以預擴增產物稀釋30倍為模版、dNTP (10 mmol/L)1.5μL、引物(10μmol/L)1.0μL、Taq酶用量1.25 U時的擴增效果較好。利用優化體繫成功篩選齣61對可以穫得帶型豐富且重複性好的引物組閤。差異片段迴收及PCR檢測的結果錶明,優化後的cDNA-AFLP分析體繫適用于油茶相關差異基因的錶達研究。
이소과유다화보통유다적눈협급충인위재료,대영향기cDNA-AFLP반응체계적관건인자진행우화분석,사선출괄합유다적cDNA-AFLP반응체계。결과표명:분별용EASYspin Plus식물RNA쾌속제취시제합화Trizol법능성공획득고질량적유다눈협화충인총RNA;종총RNA중분리적mRNA경합성쌍련cDNA후,용한제성내절매EcoRⅠ화MseⅠ재37℃하5 h내매절완전,경5 U T4련접매16℃련접적산물희석10배후가직접용우예확증;재20μL선택성확증체계중,이예확증산물희석30배위모판、dNTP (10 mmol/L)1.5μL、인물(10μmol/L)1.0μL、Taq매용량1.25 U시적확증효과교호。이용우화체계성공사선출61대가이획득대형봉부차중복성호적인물조합。차이편단회수급PCR검측적결과표명,우화후적cDNA-AFLP분석체계괄용우유다상관차이기인적표체연구。
Using the te nd er leaf, kernel of Camellia meiocarpa and C. oleifera as materials, a suitable cDNA-AFLP reaction system for C. meiocarpa and C. oleifera was established after optimizing several key factors, inc-luding enzyme digestion system, pre-amplification and selective amplification, which may affect cDNA-AFLP analysis. The results showed that using the quick extraction kit of EASYspin Plus plant RNA and Trizol method could extract ideal total RNA from the tender leaf and kernel of C. meiocarpa and C. oleifera, respectively. After mRNA was isolated from total RNA, it was synthesized into double-stranded cDNA. And then the double-stranded cDNA was digested completely by the combination of restriction endonuclease EcoRⅠand MseⅠat 37℃for 5 hours. The digested product was connected with 5 U T4 ligase at 16℃ for an overnight. Then reducible pre-amplification result was obtained when ligation product were diluted by 10 times for pre-amplification. And the optimal conditions for a 20 μL volume of selective amplification were obtained when the template of preexp-anded product diluted by 30 times and with dNTP (10 mmol/L) 1.5μL, primer (10μmol/L) 1.0μL, Taq enzyme 1.25 U. Moreover, 61 out of 210 primer pairs, producing repeatable and prolific bands, were successfully selected by this improved method. According to the PCR detected results of recovery different gene fragment, the opt-imized cDNA-AFLP technique was suitable for further expression study on the differential gene in C. oleifera.