山茶花CjAPL1基因正义表达载体的构建及对拟南芥的转化分析

Sense Expression Vector Construction and Analysis of Transgenic Arabidopsis thaliana with CjAPL1 Gene from Camellia japonica

为研究山茶花CjAPL1基因对于重瓣花形成的作用,构建了pCAMBIA1300-CjAPL1正义植物表达载体,酶切结果显示,CjAPL1基因正向插入pCAMBIA1300的启动子和终止子之间,表明CjAPL1植物表达载体构建成功。将pCAMBIA1300-CjAPL1采用农杆菌介导的花序浸染法转化拟南芥,获得转基因阳性植株65株。随机挑选表型变异明显的3株进行PCR扩增都得到了目的条带,Southern杂交鉴定进一步确认为转基因阳性植株。同时荧光定量检测到在转基因植株中CjAPL1基因表现出较对照显著提高6~25倍的表达量。转基因阳性植株表型变异明显,花柱和雄蕊数相比野生型各增加1枚和1~4枚,萼片边缘发育成白色的瓣化状,表明在拟南芥中过量表达CjAPL1基因可以引起花器官形态的变异和数量的变化,因此该基因在花器官形成和重瓣花发育中具有显著的调控功能。

In order to investigate the gene function of CjAPL1,a homolog of AP1 from Camellia japonica,during double flower formation,pCAMBIA1300-CjAPL1 sense expression vector was constructed. The restriction enzyme digestion result showed that CjAPL1 gene was correctly inserted into pCAMBIA1300 sites between 35S promoter and terminator,which indicated that an ectopic expression vector had been successfully constructed. The pCAMBIA1300-CjAPL1 construct was transformed into Arabidopsis thaliana by inflorescence soaking mediated with Agrobacterium,and 65 independent transgenic plants were obtained. Three randomly selected positive plants with obvious phenotypic changes were confirmed by PCR amplification and Southern blotting,and high expression levels of CjAPL1 in transgenic plants were also detected by the Real time-PCR analysis. Compared with WT plants,obvious phenotypic changes including increases of 1 style and 1–4 stamens,sepals edge developed petaloid morphology with white characteristics,and early flowering,were observed. These results showed that overexpression of CjAPL1 gene could cause the change of floral organ morphology and number,and therefore indicated that CjAPL1 could play notable functions during floral organ formation and double flower development.