菊花DgLsL基因RNAi表达载体的构建及遗传转化

Construction of RNAi Expression Vector of DgLsL Gene and Genetic Transformation of Chrysanthemum 'Jinba'

培育少侧枝的标准切花菊品种具有重要的实际意义。本研究利用RNAi的方法抑制菊花侧枝发育相关基因DgLsL基因的表达,以期得到侧枝生长受抑制的转基因植株。从切花菊‘神马’基因组DNA中克隆侧枝发育相关基因DgLsL基因,经过测序分析,将一个413bp长的保守片段连接到植物表达载体pBI121和pFGC5941,构建植物RNAi载体pBI121-R2,并运用农杆菌介导法将其导入切花菊‘神马’。结果:PCR得到的DgLsL基因片段测序后与GenBank上的DgLsL基因比对,DNA序列一致性为99.4%;构建的RNAi表达载体经过PCR和酶切证实外源基因的正确插入;转化后得到了18株抗性植株,PCR结果显示,12株抗性植株为阳性。本试验通过将DgLsL基因干扰片段导入切花菊,获得了转基因植株,以此为基础可进一步选育少侧枝的切花菊品种。

The breeding ofbranchless varieties has important practical value for standard cut chrysanthemum. In this study, DgLsL gene of chrysanthemum, which was related to branch development, was inhibited with the method of RNAi to get branchless transgenic chrysanthemum. DgLsL gene was cloned from cut chrysanthemum ＇Jinba＇ genomic DNA. After sequencing analysis, a 413 bp conservative fragment of this gene was inserted into the plant expression vector pBI121 and pFGC5941 to construct a plant RNA interference expression vector pBI121-R2. Then the vector pBI121-R2 was introduced into ＇Jinba＇ genome by Agrobacterium-mediated transformation. The DgLsL gene fragment obtained by PCR was sequenced, and the DNA sequence indentity was 99.4% with DgLsL gene in GenBank. The correct insertion of the exogenous gene fragments in the vectors was confirmed by PCR and restriction enzyme digestion. After transformation 18 putative transgenic plants were obtained, which 12 of them were confirmed by PCR.