紫花苜蓿蓝光受体CRY2B过表达载体构建及转化

Construction of Blue Light Receptor CRY2B over Expression Vector and Genetic Transformation in Medicago Sativa

短日照是影响苜蓿秋眠性的主要因素,即光周期效应。根据转录组测序的结果,对主要的蓝光受体隐花色素CYR2 B基因和苜蓿秋眠性之间关系的研究可以从光受体途径来揭示苜蓿秋眠性调控机制,本试验首先克隆紫花苜蓿1级品种‘驯鹿'隐花色素CRY2B基因的完整开放阅读框(696~2553 bp),设计1对含有酶切位点的特异性引物CRY2 B-F,CRY2 B-R,通过多次酶切和连接,将克隆目的片段连接到植物表达载体p CAMBIA3301上得到重组载体p CAMBIA3301-CRY2B,成功地构建了紫花苜蓿CRY2B的过表达载体,并利用农杆菌介导p CAMBIA3301-CRY2 B成功转化苜蓿植株,为进一步研究CRY2 B调控苜蓿秋眠性分子机制奠定基础。

The short-day is one of the main factors affected alfalfa fall dormancy,which is called as the photoperiodic effect. Based on the results of alfalfa transcriptome sequencing,the relationship between the main photoreceptorsCRY2B gene and alfalfa fall dormancy may reveal the regulation mechanism of alfalfa fall dormancy. In this experiment,cryptochrome gene--CRY2B of Reindeer（the variety of fall dormancy class NO. 1） with complete open reading frame（696 ~ 2553 bp） was cloned. For the design of specific primers with the restriction sites（CRY2B-F and CRY2B-R）,the cloned fragment was connected to the plant expression vector p CAMBIA3301 to get a recombinant vector p CAMBIA3301-CRY2B after enzymes digestion,clean up and ligation. Agrobacterium-mediated genetic transformation using alfalfa cotyledonary node was explored,it laid the foundation for the further research of CRY2B regulating fall dormancy molecular mechanism.