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为了探究LEC1基因对棉花体细胞胚胎发生的影响,本研究利用地塞米松(DEX)诱导型表达载体p INDEX3,通过农杆菌分别介导大叶落地生根短缺的LEC1(Kd LEC1)在新海15号下胚轴以及棉花的两个LEC1基因家族(Gb LEC1A,Gb LEC1B)和Kd LEC1在新陆早33号胚性愈伤组织中的遗传转化,体胚诱导及再生过程,获得Kd LEC1、Gb LEC1A、Gb LEC1B转基因植株。同时以Kd LEC1转基因新海15号胚性愈伤组织系为试材,分别进行DEX和无DEX的悬浮诱导处理,实时定量PCR分析Kd LEC1的相对表达量。研究表明:经为期13个月的植株再生过程和PCR鉴定,获得了两棵Kd LEC1转基因新海15再生植株;分别获得了一棵PCR鉴定正确的Kd LEC1、Gb LEC1A、Gb LEC1B转基因新陆早33再生植株,其再生周期约为10个月。实时定量PCR分析显示,Kd LEC1的相对表达量在2.5倍和23倍之间波动,表明p INDEX3可以在不同水平诱导外源基因Kd LEC1的表达。本研究旨在为LEC1在棉花体胚发生过程中的功能研究提供良好的材料,从而为棉花种质创新提供理论指导。
In order to explore the effect of LEC1 gene on somatic embryogenesis in cotton, we used the inducible expression vector p INDEX3 of dexamethasone (DEX) to induce LEC1 (Kd LEC1) Hypocotyls and cotton two LEC1 gene families (Gb LEC1A, Gb LEC1B) and Kd LEC1 Xinluza 33 embryogenic callus in the genetic transformation, somatic embryo induction and regeneration process to obtain Kd LEC1, Gb LEC1A , Gb LEC1B transgenic plants. At the same time, the embryogenic callus of Kd LEC1 transgenic Xinhai 15 was used as experimental materials, and DEX and DEX-free suspension induction treatment were carried out respectively. The relative expression of Kd LEC1 was analyzed by real-time quantitative PCR. The results showed that two Kd LEC1 transgenic Xinhai 15 regenerated plants were obtained after 13-month plant regeneration and PCR identification. A PCR-positive Kd LEC1, Gb LEC1A and Gb LEC1B transgenic lines, Plant regeneration, the regeneration cycle is about 10 months. Real-time quantitative PCR analysis showed that the relative expression of Kd LEC1 fluctuated between 2.5 times and 23 times, indicating that p INDEX3 can induce the expression of the foreign gene Kd LEC1 at different levels. The purpose of this study is to provide a good material for the functional study of LEC1 in the process of somatic embryogenesis of cotton and to provide theoretical guidance for cotton germplasm innovation.