紫花苜蓿光敏色素B基因片段克隆及RNA干扰表达载体的构建

doi:10.3724/SP.J.1006.2011.00374

摘要/Abstract

摘要： 根据紫花苜蓿2级标准品种Vernal PhyB基因的序列(GenBank登录号为GQ379903.1)，设计2对含有酶切位点的特异性引物F1/R1和F2/R2，以紫花苜蓿总RNA为模板，通过RT-PCR法扩增得到PhyB基因正向、反向目的片段，将片段连接到pGEM-T Easy载体上得到重组载体pGEMB-1和pGEMB-2，再以中间载体pHANNIBAL和植物双元表达载体pART27为基础，通过多次酶切和连接，成功地构建了紫花苜蓿PhyB的RNAi表达载体。为进一步研究光敏色素B与苜蓿秋眠性之间的关系奠定了基础。

关键词: 紫花苜蓿, 秋眠性, 光受体, 光敏色素, RNA干扰

Abstract: The short day is one of the main factors affecting alfalfa fall dormancy, which is called as the photoperiodic effect. Study on the relationship between the main photoreceptors-PhyB gene and alfalfa fall dormancy may reveal the regulation mechanism of alfalfa fall dormancy radically, and provide a scientific reference for the application of alfalfa varieties differing in fall dormancy in forage production. The objective of this study was to establish an RNAi expression vector harboring PhyB gene of Medicago sativa. Two pairs of specific primers containing different enzyme sites were designed on the basis of PhyB gene sequence of alfalfa variety “Vernal” (GenBank accession number: GQ379903.1). With the template of total RNA, positive sense strand and antisense strand were amplified by RT-PCR and cloned into pGEM-T Easy vector to obtain recombinant vectors pGEMB-1 and pGEMB-2, then based on the intermediate vector pHANNIBAL and the plant binary expression vector pART27, we constructed the RNAi expression vector pART27-RNAi containing a hairpin structure by many times of enzyme digestion and connection. The results provide a foundation for further studying the relationship between alfalfa dormancy and PhyB.

Key words: Medicago sativa, Fall dormancy, Photoreceptor, Phytochrome, RNA interference

朱见明, 严学兵, 史莹华, 王成章. 紫花苜蓿光敏色素B基因片段克隆及RNA干扰表达载体的构建[J]. 作物学报, 2011, 37(02): 374-379.

SHU Jian-Meng, YAN Hua-Bing, SHI Ying-Hua, WANG Cheng-Zhang. Cloning of Medicago sativa Phychrome B cDNA and Establishment of Its RNA Interference Expression Vector[J]. Acta Agron Sin, 2011, 37(02): 374-379.

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