pBAR载体构建及其转化罗汉果的分析

Construction of pBAR Vector and Analysis of Its Transformation into Siraitia grosvenorii

为了创制可免除人工授粉的转基因罗汉果雌性品系,利用植物双元表达载体pBI121-Gus,构建幼果实特异启动子2A11与单性结实相关基因rolB的嵌合基因表达载体pBAR (pBI121-2A11-rolB)。以罗汉果雌株叶盘为材料,以EHA105农杆菌介导遗传转化,经PCR扩增,鉴定出阳性植株,对阳性雌株进行扩繁、生根,最后移栽大田,并观察转基因植株的单性结实性状表现。结果显示,成功构建了单性结实相关基因的pBAR嵌合双元表达载体;转化感受态根癌农杆菌EHA105后,进一步转化雌株叶片,经过对叶片分化苗的检测,得到7株阳性植株,转化率为14.29%。为提高成活率,扩繁7株阳性植株,获得37株转基因单性结实植株株系,其中有8株正常开花,占总数的21.62%,正常开花的植株,未经人工授粉,发育成幼果,表现出单性结实性状。本实验在克隆单性结实相关基因和果实特异启动子的基础上,构建嵌合基因载体,通过根癌农杆菌介导的叶盘转化法,获得了转基因罗汉果单性结实雌株系,为后续研究罗汉果单性结实性的遗传、生理、品种综合改良和深入利用提供了基础。

In order to create a transgenic Siraitia grosvenorii female line that can be exempted from artificial pollination, the chimeric gene expression vector p BAR(ie., p BI121-2 A11-rolB) of the young fruit-specific promoter2 A11 and the parthenocarpy-associated gene rolB was constructed by the plant binary expression vector pBI121-Gus. The female leaf disc of Siraitia grosvenorii was used as the material, and the genetic transformation was mediated by EHA105 Agrobacterium tumefaciens. The positive plants were identified by PCR amplification.The positive female plants were expanded, rooted, finally transplanted into the field, and the parthenocarpic characteristics of the transgenic plants was observed. The results showed that the pBAR chimeric binary expression vector of parthenocarpy-related genes was successfully constructed. After transformation of competence Agrobacterium tumefaciens EHA105, the leaves of female plants were further transformed. After detection of leaf differentiation seedlings, 7 positive plants were obtained, the transformation rate was 14.29%. In order to improve the survival rate, 7 positive plants were expanded and 37 transgenic parthenocarpic plant lines were obtained.Among them, 8 plants were normally flowering, accounting for 21.62% of the total. Normally flowering plants developed into young fruit without artificial pollination, showing parthenocarpic characteristics. In this experiment, based on the cloning of parthenocarpy-associated genes and fruit-specific promoters, a chimeric gene vector was constructed, and the transgenic Siraitia grosvenorii parthenocarpic female line was obtained by Agrobacterium tumefaciens-mediated leaf disc transformation. It would provide a basis for the follow-up study on the genetic, physiological, comprehensive improvement and in-depth utilization of the parthenocarpy of Siraitia grosvenorii.