大麦cDNA-AFLP技术体系的优化及其应用

Optimization and Application of cDNA-AFLP System in Barley

为构建适合分析大麦杂种优势的cDNA-AFLP技术体系,以4个大麦不育系、2个大麦恢复系以及按4×2配制的8个杂交种为材料,对影响大麦cDNA-AFLP技术体系的几个关键因素进行了研究。结果表明,大麦cDNA-AFLP技术按以下程序可得到多态性好的清晰条带:使用百泰克公司的TRIpure Reagent总RNA提取试剂可提取到高质量的叶片总RNA;用TaKaRa的M-MLV RTase反转录合成双链cDNA;用MseⅠ和EcoRⅠ酶切6h,再用T4连接酶15℃连接接头18h;预扩PCR反应的dNTP终浓度为0.2mmol.L-1,扩增20循环效果最好;预扩产物稀释20倍后进行选择性扩增,最终用6%的变性聚丙烯酰胺凝胶进行电泳分离条带。利用该cDNA-AFLP体系对参试杂交种及其亲本的谱带类型进行了分析,亲本与杂种之间共扩增出7种类型的谱带,分别是共同表达型(P1P2F1)、P1表达型(P1)、P2表达型(P2)、F1表达型(F1)、P2F1表达型(P2F1)、P1F1表达型(P1F1)和P1P2表达型(P1P2)。其中差异谱带类型可分为四种表达模式:(1)单亲显性表达型,包括P2F1表达(P2F1)和P1F1表达(P1F1);(2)单亲沉默表达型,包括仅P1表达(P1)和仅P2表达(P2);(3)杂种上调表达型,即仅F1表达(F1);(4)杂种下调表达型,即仅P1P2表达(P1P2)。

Eight hybrids were produced by four barley male sterile lines and two barley restorer lines in a 4X2 design. Then the parents and their hybrids were used as materials to establish the cDNA-AFLP system for barley. And the key factors affect the system were studied in this paper. The result indica- ted that a distinct electrophoretogram of cDNA-AFLP could be displayed by the below procedures. The TRIpure Reagent of Biteke was used to extract the total RNA from the leaf quickly and efficient- ly. Then the double strand cDNA was synthesized by the M-MI.V-RTase of TaKaRa and digested by Mse Ⅰ and EcoR Ⅰ at 37℃ for 6 hours in one step. The digested product was ligated to the adapter by T4 ligase at 15℃ for 18 hours. The optimal concentration of the dNTP was 0.2mM and the optimal cycle of the pre-amplification was 20 for the pre-amplification. Then the pre-amplified product was di- luted by 20 times as the template of selective amplification. The product of selective amplification was checked by the 6 ~//00 PAGE. The band types of the hybrids and their parents were also analyzed by the cDNA-AFI.P system. Seven types of bands were obtained between parents and their hybrids, which were co-expression type （P1P2F1）, only P1 express type （Pl）, only P2 express （P2）, only F1 express （F1）, rized P2F1 type （PzF1） , P1F1 as 4 expression patterns, type （P1F1） which were single silence expression type including lence expression type P1P2. and P1P2 type （P1 P2）. The difference bands were summa- single dominant expression type including P2 F1 and P1 F2, and P2, hybrid enhanced expression type F1 and hybrid si-