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作者	单位
张新忠，吕亮杰，吕超，许如根	（扬州大学江苏省作物遗传生理重点实验室/扬州大学大麦研究所,江苏扬州 225009）

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中文摘要:

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6 h，再用T4连接酶15℃连接接头18 h；预扩PCR反应的dNTP终浓度为0.2 mmol·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）。

英文摘要:

Eight hybrids were produced by four barley male sterile lines and two barley restorer lines in a 4×2 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 indicated 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 efficiently. Then the double strand cDNA was synthesized by the M MLV RTase of TaKaRa and digested by MseⅠand EcoRⅠat 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 diluted by 20 times as the template of selective amplification. The product of selective amplification was checked by the 6% PAGE. The band types of the hybrids and their parents were also analyzed by the cDNA AFLP system. Seven types of bands were obtained between parents and their hybrids, which were co expression type (P₁P₂F₁), only P₁express type (P₁), only P₂ express (P₂), only F₁express (F₁), P₂F₁type (P₂F₁), P₁F₁type (P₁F₁) and P₁P₂ type (P₁P₂). The difference bands were summarized as 4 expression patterns, which were single dominant expression type including P₂F₁and P₁F₁, single silence expression type including P₁and P₂, hybrid enhanced expression type F₁and hybrid silence expression type P₁P₂.

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