韧皮部特异启动子CoYMV调控下CCoAOMT基因改良黄麻纤维品质的研究

Jute is one of the most important fiber crops in the world, with the planting area and yield almost equal to that of cotton. Jute fibers exhibit a characteristically good moisture absorption performance, rapid water loss capacity and easy degradation. The quality of jute fiber is very poor mainly due to the higher lignin content, which seriously restrict the application of jute fiber in the textile industry and multi-utilization. In this study, we would isolate the full-length cDNA of the Caffeoyl CoA O-methyltransferase (CCoAOMT) gene from jute , by homologous cloning (primers were designed according to the sequence of CCoAOMT gene of other plants) and modified RACE techniques. Differential spatial and temporal expression of CCoAOMT gene would be revealed by Real-time PCR analysis. At the same time, Phloem-Specific Promoter CoYMV also would be cloned, which activity would be testified using histochcmical localization of GUS activity through trangenic tobacco plants. Moreover, the RNAi vector including both CoYMV and CCoAOMT gene would be constructed and integrated into tobacco plants to verify their function. Then, the vector would be integrated into jute plants using agrobacterium-mediated transformation technique. The transgenic plants would be screened from DNA, RNA and protein level. The characteristics as to fiber quality, such as lignin content, fiber fineness, fiber strength and anthracnose risistance would also be measured, so as to analysis the function of improving fiber quality by suppression of CCoAOMT gene through phloem-specific promoter CoYMV . The results could provide an important basis for the application of CCoAOMT gene in the improvement of jute fiber quality. This study has important significance to improve the level of jute breeding, as well as to improve economic benefits of jute planting.

黄麻是世界上重要的纤维作物，其纤维具有透气性好、吸湿性强、易降解等特性。然而，黄麻纤维中木质素含量较高而导致纤维粗、硬，品质不佳，限制了其在纺织等工业领域的应用。本研究拟采用同源克隆技术，克隆黄麻的木质素合成关键酶基因CCoAOMT，采用Real-time PCR技术分析其在黄麻中的表达情况，并将其转化至烟草中进行功能验证；克隆韧皮部组织特异启动子CoYMV，构建其驱动GUS基因的表达载体，转化烟草植株，分析其组织特异表达活性；构建同时包含特异启动子CoYMV和目的基因CCoAOMT的干扰表达载体，以农杆菌介导的方法将其转化至黄麻植株中，分别从DNA、RNA和蛋白质水平对转化植株进行验证；通过测定转基因黄麻植株中的木质素含量、纤维支数、纤维强力等纤维品质性状和抗倒伏、抗病性等抗逆性状，阐明在韧皮部特异启动子CoYMV作用下，CCoAOMT基因在降低黄麻木质素含量、改良黄麻纤维品质上的功能。

根据本项目研究计划的进度和内容安排,项目组开展的研究内容和取得的研究结果如下:.1.进行了黄麻CCoAOMT基因全长序列的克隆，选取了不同物种的CCoAOMT的氨基酸序列,通过分析发现，黄麻CCoAOMT氨基酸与米芹的亲缘关系最近，与马铃薯、葡萄的亲缘关系最远。CCoAOMT基因在黄麻各组织中都有表达，其中麻骨中表达量最高，其次是根、茎皮和叶片中。这与CCoAOMT基因在木质化程度较高的维管束和厚壁组织中高效表达有关。.2.开展了韧皮部特异启动子CoYMV的组织特异表达活性分析、CCoAOMT基因过表达载体构建、转化拟南芥及功能验证等工作。利用竹节花黄班驳病毒启动子(CoYMV)驱动GUS基因的表达载体转化模式植物烟草，结果表明CoYMV驱动GUS基因表达活性与35S相比没有明显差异，更重要的是CoYMV能驱动GUS基因在烟草韧皮部中特异表达，结果表明了该启动子具有韧皮部组织特异性，可用于下一步木质素基因的定点抑制。项目组利用35S驱动CCoAOMT基因在拟南芥中过量表达，转基因拟南芥木质素含量与对照相比明显增加，整个植株表现为硬度和高度增加，结果表明黄麻CCoAOMT基因与纤维素合成及木质化进程密切相关，为进一步利用RNA干扰该基因改良黄麻纤维品质提供了新的途径。.3. 构建了同时包含CoYMV与CCoAOMT基因的RNA干扰载体和人工定向沉默载体；通过农杆菌介导的方法将其转化至黄麻品种“179”植株中；对转基因植株进行了潮霉素筛选、PCR和Southernblot验证，结果表明外源基因已整合进入黄麻基因组。测定了转化黄麻植株纤维中木质素含量和纤维品质性状及抗逆性状，结果表明转化植株木质素含量降低，纤维品质提高，抗逆性不变，表明在特异启动子CoYMV调控下，CCoAOMT基因能够改良黄麻纤维品质。.综上所述，本项目的开展为选育低木质素含量的优异黄麻新品种奠定了理论基础，同时对改良黄麻纤维品质、扩大黄麻在工业领域的应用及提高黄麻产业链的经济效益等具有较重要意义。