Construction of a set of small and useful binary Ti plasmid vectors for plant transformation

构建一套小型且有用的二元 Ti 质粒载体用于植物转化

• 批准号: 16580007
• 负责人: ICHIKAWA Hiroaki
• 金额: $ 2.37万
• 依托单位: National Institute of Agrobiological Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16580007/
• 关键词: Binary vector; Plant transformation; Agrobacterium; Plant biotechnology; Plant molecular biology; T-DNA; pVS1; Plasmid stability; 植物形質転換ベクター; アグロバテリウム; 遺伝子組換え植物; トランスジェニック植物

1.By appropriately combining replication protein repA [3 types : high-copy type, wild-type, and low-copy type (LC)] and staA or par fragments conferring plasmid stability upon Agrobacteria, very small plasmid backbones enabling replication in both E.coli and Agrobacterium were constructed. Then, Pnos::Hyg^R marker and P35S::gusA reporter cassettes were introduced into the shuttle vectors to generate five mini-binary vectors. The novel binary vectors were very small (8.6〜9.65 kb) and carry 4 multicloning sites between Hyg^R and gusA genes. In addition, I got three Gateway-type binary vectors by substituting gusA with the Gateway cassette.2.The abovementioned five mini-binary vectors, after introducing into Agrobacterium, were transformed into rice based on tissue culture and into Arabidopsis by using floral-dip method (without tissue culture). The transformation frequencies were high enough to obtain many transgenic plants from both rice and Arabidopsis. Southern blot analysis indicated that relatively low-copy numbers (one to three) of T-DNAs were integrated into transgenic rice plants, especially when transformed with pSTARH301G carrying repA-LC + staA.3.Rice acetolactate synthase (ALS) gene bearing two different point mutations in the coding sequence (CDS), which confers herbicide tolerance upon rice plants, was further changed to delete a single HindIII site in the CDS. This fragment (mALS) was subcloned along with 1.37 or 0.56 kb promoter regions. The two mALS cassettes were individually introduced into the two mini-binary vectors, pSTARH301G and pPARH5O1G, to generate four different vectors harboring the long or short mALS herbicide-tolerant marker gene. All the four vectors were introduced into rice via Agrobacterium, and gave good transformation frequencies, indicating even mALS with short promoter could work excellently in rice transformation.

1.通过适当组合复制蛋白repA[3种类型：高拷贝型、野生型和低拷贝型(LC)]和赋予农杆菌质粒稳定性的staA或par片段，构建了能够在大肠杆菌和农杆菌中复制的非常小的质粒主链。然后，将 Pnos::Hyg^R 标记和 P35S::gusA 报告基因盒引入穿梭载体中，生成五个迷你二元载体。新型双元载体非常小（8.6〜9.65 kb），并且在Hyg^R和gusA基因之间携带4个多克隆位点。另外，将Gateway盒替换gusA，得到了3个Gateway型双元载体。2、将上述5个微型双元载体导入农杆菌后，基于组织培养转化水稻，并采用浸花法（无组织培养）转化拟南芥。转化频率足够高，足以从水稻和拟南芥中获得许多转基因植物。 Southern印迹分析表明，相对低拷贝数（1到3个）的T-DNA被整合到转基因水稻植物中，特别是当用携带repA-LC + staA.3的pSTARH301G转化时。 编码序列（CDS）中带有两个不同点突变的水稻乙酰乳酸合酶（ALS）基因，赋予水稻植物除草剂耐受性，进一步改变以删除单个HindIII CDS 中的站点。该片段 (mALS) 与 1.37 或 0.56 kb 启动子区域一起亚克隆。将两个mALS盒分别引入两个微型二元载体pSTARH301G和pPARH5O1G中，以产生四种不同的载体，其含有长或短mALS除草剂耐受标记基因。所有四种载体均通过农杆菌导入水稻，并给出了良好的转化频率，表明即使具有短启动子的mALS也能在水稻转化中发挥出色的作用。