牡丹ACC氧化酶基因片段的克隆及其反义表达载体的构建

利用CTAB改良法提取牡丹洛阳红花瓣总RNA,通过反转录酶和设计的ACC氧化酶基因特异引物合成709bp目的片段,进行测序和比对,以确定目的片段的正确性。在709bpACC氧化酶基因片段和pBI121植物表达载体上选择合适的酶切位点,进行XbaⅠ和SmaⅠ双酶切。将709bp片段上切下的510bp片段反向插入pBI121植物表达载体35S启动子后面,构建成含510bp牡丹ACC氧化酶基因反义表达载体。最后采用电击法转化进感受态农杆菌GV3101,以备用于花粉管通道法转化牡丹。

Cloning of ACC Oxidase Gene of Tree Peony and the Construction of Antisense Expression Vector

The total RNA was extracted from the petals of Luoyanghong-a cultivar of tree peony,and 709bp objective fragment was synthesized by the actions of reverse transcriptase and ACC oxidase gene specific primer,then sequenced and compared it to confirm the correctness. The suitable restriction sites were choosed in the 709bp ACC oxidase gene fragment and pBI121 plant expression vector,then carried out XbaⅠand SmaⅠdigestion. The 510bp fragment cutting from the 709bp fragment was reversely inserted into pBI121 plant expression vector behind 35S promoter,constructing of antisense expression vector containing 510bp tree peony ACC oxidase gene. Finally the pBI121 plasmid containing the 510bp objective fragment was transformed into the permissive agrobacterium GV3101 by the method of electric shock,to prepare for genetic transformation tree peony by the method of the pollen tube pathway.