【作者】 郑武；

【导师】 李付广；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2014， 博士

【摘要】 植物体细胞胚胎发生是离体研究合子胚发育机制的理想模型。棉花转基因技术中，基于体细胞胚胎发生的农杆菌介导法是应用最广泛的技术之一。目前棉花体细胞胚胎发生依然比较困难，其中愈伤组织如何高效快速的分化为胚性愈伤组织是主要技术瓶颈。只有少数品种可以实现高效快速的分化，大多数品种表现为难分化或不分化。这一难题严重限制了转基因技术在更广泛品种上的应用。因此寻找控制棉花体细胞胚胎发生过程分化的关键基因并研究其分子机制就显得尤为重要。本实验从转基因角度出发，通过农杆菌介导法将目的基因转入棉花以验证目的基因在棉花愈伤组织分化为胚性愈伤组织时期的功能，并取得了以下实验结果：(1)利用实验室已经构建好的抑制性消减杂交（SSH）文库数据，寻找到在胚性愈伤组织时期表达量相对较高的目的基因GhSERK1和GhMAPK16，并通过电子克隆和基因组数据克隆到编码区。将这些基因构建过表达载体转化陆地棉品种CRI24，结果发现GhSERK1确实在胚性愈伤组织时期的表达量呈现高峰；过表达GhSERK1不能从本质上提高愈伤组织分化率但可以加快愈伤组织形成胚性愈伤组织的进程。而GhMAPK16在胚性愈伤组织时期的表达量并没有呈现高峰，而且过表达该基因也没有任何表型。(2)利用已证明能促进拟南芥胚性愈伤组织形成的基因AtWuschel，构建过表达载体转化陆地棉难分化材料CRI12。结果发现CRI12的分化率从0.61%提高到47.75%，极大的提高了该材料的愈伤组织分化率。在此基础上，我们进一步探讨了该基因促进体细胞胚胎发生的分子机制，结果发现AtWuschel极有可能是通过激活生长素信号通路进而使得生长素下游的体细胞胚胎发生潜能基因GhLEC1、GhLEC2和GhFUS3得到激活来行使其功能的。为了进一步验证GhLEC1、GhLEC2和GhFUS3的功能，我们构建了一个同时干涉GhLEC1、GhLEC2和GhFUS3的干涉载体，并转化高分化率材料CRI24，结果发现CRI24的分化能力也受到严重抑制。(3)为了能更广泛的筛选到控制体细胞胚胎发生的关键基因，构建了愈伤组织到胚性愈伤组织全生育期的过表达的cDNA全长均一化文库（全长cDNA质粒穿梭文库），为以后将整个文库通过农杆菌介导法转化棉花来大规模筛选关键基因奠定了基础。综上所述，候选的三个基因中，AtWuschel能显著提高愈伤组织的分化率。生长素和细胞分裂素信号通路基因及其下游基因GhLEC1、GhLEC2和GhFUS3的表达量在高分化材料愈伤组织（CRI24）和难分化或不分化材料（CRI12、CRI41、Lu28）的愈伤组织间存在明显差异。而AtWuschel可能正是通过调节这些通路和下游基因才使得难分化材料CRI12的分化率提高。这表明生长素和细胞分裂信号通路以及GhLEC1、GhLEC2和GhFUS3在控制愈伤组织分化能力上起着非常重要的作用。更多还原

【Abstract】 Plant somatic embryogenesis can serve as model systems to study zygote embryo development invitro. Among all the cotton transgenic techonologies, somatic embryogenesis-dependent agrobacteriummediated transformation was widely used. So far, cotton somatic embryogenesis was still very difficμlt,the low differentiation frequency from callus to embryogenic callus was a main limiting factor. Onlyfew varieties have a high differentiation frequency, most of varieties are hard to differentiate or not. Theresult is that transgenic techonology could not be applied in most of cotton varieties. Therefore, thefuture work is to found the key genes controlling callus differentiation and investigate the molecularmechamism. In our study, we utilized agrobacterium-dependent transgenic technology to determine thetarget genes function in controlling embryogenic callus induction. In conclusion, we obtained thefollowing results:(1)Through the analysis of suppression subtractive hybridization data, we found the target genesGhSERK1and GhMAPK16, which had relatively higher expression level in embryogenic callus. Thenwe ulitized electronic cloning and genome data to obtain the CDS sequence.we construct theover-expression vector to transform CRI24, the result was that GhSERK1had a higher expression levelin embryogenic callus, which shorten the process of callus developing into embryogenic callus and hadno significant influence on the differentiation frequency.while GhMAPK16did not had a higherexpression level in embryogenic callus, its overexpression had no effects on callus differentiation.(2) AtWuschel could promote embryogenic callus formation in Arabidopsis, we constructedAtWuschel overexpression vector to transform CRI12which was hard to differentiate. As a result, thedifferentiation frequency of callus was increased from0.61to47.75percent. To investigate themolecular mechanism, we took some experiments. Finally we found that it was possible to activate theauxin signal and cytokinin pathway to induce the downstream gene expression. These downstreamgenes including GhLEC1, GhLEC2and GhFUS3were closely related with somatic embryogenesis. Toinvestigate the function of GhLEC1, GhLEC2and GhFUS3, we constructed the interference vector tosilence these three genes and then transformed the interference vector to CRI24, the result was that thedifferentiation ability of CRI24was seriously inhibited.(3)In order to obtain the key genes controlling somatic embryogenesis, we constructed ahomogenization full-length cDNA library from callus to embryogenic callus. It lays a foundation fortransforming the full-length cDNA library to cotton and obtaining the key target genes controllingsomatic embryogenesis.In conclusion, we found that AtWuschel could increase callus differentiation frequency. Genesinvolved in auxin signal pathway, cytokinin signal pathway and the dowenstream genes GhLEC1,GhLEC2and GhFUS3had differential expression level between CRI24and CRI12, CRI41, Lu28.CRI24had a high differentiation frequency, while CRI12, CRI41and Lu28were hard to differentiate.According to the mentioned above, AtWuschel may regμlate the genes involved in auxin and cytokinin signal pathway and the downstream genes to increase the differentiation frequency of CRI12. Theseresults showed that genes involved in auxin and cytokinin signal pathway and the downstream genesGhLEC1, GhLEC2and GhFUS3may play critical roles in controlling callus differentiation.更多还原