用单性结实基因2A11-iaaM转化罗汉果的研究

Transformation of Siraitia grosvenorii with 2A11-iaaM gene

为了实现罗汉果生产中免除人工授粉和果实无籽化,该研究利用pBI121-Gus构建果实特异启动子2A11与生长素合成相关基因iaaM的嵌合基因(2A11-iaaM)过量表达载体,以罗汉果雌株叶盘为材料,采用农杆菌介导法建立罗汉果高效遗传转化体系,转化和创制单性结实罗汉果种质,通过基因特异引物对的PCR扩增,初步检测出转基因阳性植株,将之移栽大田,观察转基因植株的单性结实性的表现。结果表明:构建罗汉果单性结实性相关的pBAI-Gus植物双元表达载体获得成功;建立了农杆菌介导的罗汉果叶盘遗传转化优化体系,即农杆菌菌液OD_(600)值为0.3～0.5,侵染10 min,最优选择培养基为MS+TDZ 0.7 mg·L^(-1)+IBA 0.5 mg·L^(-1)+Kan 5 mg·L^(-1)+Cef 300 mg·L^(-1);经PCR鉴定共获得4株转基因阳性雌株;将阳性植株扩繁后移栽田间,经田间调查发现,24株阳性扩繁植株中有5株正常开花,占总植株数的20.8%,且其子房未经人工授粉发育成幼果,表现单性结实性。在载体构建和农杆菌介导的罗汉果遗传转化体系优化的基础上,将外源单性结实相关嵌合基因整合进罗汉果基因组并得到表达,为后续研究单性结实罗汉果的遗传生理,创制转基因罗汉果单性结实新种质,以及克服其产业化中需要人工授粉和无籽化提供了理论和应用基础。

To solve the seedless fruit and artificial pollination problems of Siraitia grosvenorii,we used pBI121-Gus to construct chimeric expression vector(pBI121-2A11-iaaM-Gus)with fruit specific promoter 2A11 and auxin synthesis key enzyme tryptophan monooxygenase enzyme gene iaaM.We took leaf disc of female plants as explants to study the antibiotics sensitivity,the impact of bacteriostatic in different concentrations of agrobacterium EHA105,and the different microbial infection time,to establish an efficient agrobacterium-mediated genetic transformation system of S.grosvenorii,and to create the parthenocarpy germplasm of S.grosvenorii.We also designed gene specific primers,and detected the positive plants by PCR technique and transplanted into the field,while recorded the hereditary characters of the transgenic plants.The results showed that the chimeric expression vector(pBI121-2A11-iaaM-Gus)related to parthenocarpy character of S.grosvenorii was successfully constructed,and the agrobacterium-mediated genetic transformation system of S.grosvenorii leaf disc was established.The optimal concentration of infection of agrobacterium tumefaciens was the OD 600 value 0.3-0.5,while the optimal infection time was 10 min.And the optimal selective medium was with 5 mg·L^-1 kanamycin and 300 mg·L^-1 cefotaxime sodium.PCR results showed that four female plants of S.grosvenorii were tested positive.We propagated the four positive female plants in the plant tissue culture room and transplanting the propagated 24 positive female plants to the field.Then the field investigation showed that there were five female plants flowering normally with a proportion of 20.8%,and the S.grosvenorii young fruits which had not been artificially pollinated were observed,which suggested the parthenocarpy character was expressed.The study,integrating the exogenetic parthenocarpy gene into the S.grosvenorii genome and preliminarily expressed,based on expression vector construction and the agrobacterium-mediated genetic transformation system of S.grosvenorii,provides the information for the subsequent study of the genetic physiology,the creation of the transgenosis parthenocarpy germplasm of S.grosvenorii,and solving the seedless fruit and artificial pollination problems of S.grosvenorii.