甘蔗体细胞融合育种中杂合体细胞再生植株的分子机制

Plant cell fusion breeding technology can overcome the limitations of traditional cross-breeding and modern molecular breeding by transferring large number of genes from the cytoplasm and the nucleus of target cell to newly produced cell, so that the new cell can possess and express multiple quantitative genetic traits from the cell of different cultivar. Regeneration is often the bottleneck in somatic hybridization breeding programs. In our previous studies, the fusion and breeding system of sugarcane somatic cell of ROC22 and GT28 had been established and optimized and the regenerated plants have obtained. Donor material genotype and environmental conditions have often been decisive for successful regeneration. The influence of environment on regeneration capacity is believed to be the result of interaction with genes. In view of this, the first, the different transcript expressing profile data of sugarcane cells in the stage of enzymatic hydrolysis, passivation, fusion and culture et al. were expressed by using RNA-seq techniques in this study, and the regulating network of regeneration occurring will be constructed, then the key genes response to regeneration plant occurring will be screened. Secondly the expressing character of the obtained key genes were evaluated in the process of regeneration occurring and treatment by using 6 regeneration regulating hormones, their functions will be verified by over expression and RNAi techniques. Finally, we will clone these key genes and analyze their expression in different relationship with hormone. Then we will know the molecular mechanism that how the mechanism of the regeneration sugarcane of hybrid somatic cells occurred. The knowledge obtained above contribute to regulate the regeneration plant occurring by using the key genes or pathways.

植物体细胞融合育种能打破传统杂交育种和分子育种的局限，同时具有转入细胞质和细胞核中因而获得多个数量遗传性状等特点。而杂合体细胞再生植株是体细胞融合育种的瓶颈。项目组在前期研究中，建立和优化了新台糖22号和桂糖28号甘蔗体细胞融合育种体系并获得了再生植株，发现细胞再生能力与基因型和环境有密切关系，前人认为环境的对再生能力影响是与基因互作的结果。本项目拟利用 RNA-Seq技术研究甘蔗体细胞在酶解、钝化、融合、培养等阶段基因差异表达谱，明确与杂合体细胞再生密切相关的代谢途径，筛选关键候选基因，然后利用荧光定量 PCR 技术研究候选基因在杂合体细胞再生不同阶段表达模式及相关调控激素的关系，通过过表达和基因沉默技术验证候选基因的功能，建立杂合体细胞过程中相关基因的联结关系和调控网络，研究候选基因与细胞骨架的调控关系，为今后提高体细胞融合育种效率和精准调控甘蔗杂合体细胞再生植株奠定重要的理论基础。

植物体细胞融合育种能打破传统杂交育种和分子育种的局限，而杂合体细胞再生植株是体细胞融合育种的瓶颈，环境的对再生能力影响是与基因互作的结果。前期研究已经建立了体细胞酶解、钝化、融合和培养体系，获得了高活力（80-90%）和高分裂频率的原生质体作为研究材料。本研究分别对酶解、钝化、融合和培养各阶段进行与细胞再生相关的分子机理研究。本课题首先优化甘蔗原生质体RNA提取条件和方法，然后对甘蔗幼叶酶解过程进行转录组学分析，对酶解、钝化、融合、培养过程进行细胞学（细胞膜、细胞核、微管骨架）、生理学、蛋白质组学分析，克隆ScWOX8和PSK基因并进行相关的遗传转化研究，进一步利用抗坏血酸和激素及渗透基因调控甘蔗原生质体再生，建立甘蔗原生质体酶解、钝化、融合、培养的调控网络。.重要研究结果如下：.1.酶解、钝化、融合诱导甘蔗原生质体的渗透和氧化应激，从而改变生物能量代谢、细胞壁合成和细胞周期调节相关蛋白质的表达和微管骨架的解聚；降低胁迫相关酶活性，从而阻碍氧化产物(O2-)的消除，改变原生质体再生相关基因（ScWOX8, PSK,GAUT,CESA,Cyclin A,Cyclin B,Cyclin D3,cdc2,Aux/IAA）的表达。.2.激素通过调控细胞壁合成（GAUT、CESA）、细胞周期调控（cyclin B、cyclin A）、细胞增殖（PSK）、生长素和细胞分裂素信号转导途径（Aux/IAA、cyclin D3、cdc2）等与杂合体细胞再生相关的关键基因表达，从而在甘蔗杂合体细胞再生中起至关重要的作用。.3.ScWOX8基因，亚细胞定位定位于细胞核，应对甘蔗原生质体再生困难可以考虑在培养过程中通过改变培养基的激素和渗透压来影响ScWOX8基因的表达从而达到促进甘蔗原生质体持续分裂的目的。.4.添加0.5 mM ASA能通过提高相关抗氧化酶基因的表达，增加抗氧化酶活性，防止氧化胁迫，促进细胞周期运转，促进细胞壁再生和细胞分裂。.5.构建了利用分子标记和抗体标记检测酶解、钝化、融合体系的假设模型，并提供了相关参数。.本研究结果为研究甘蔗体细胞融合育种杂合体细胞再生的分子、生理和细胞学机制提供了新的见解及技术支撑。