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.通过适当地组合复制蛋白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骨架。然后，将Pnos：：Hyg^R标记和P35 S：：gusA报告盒引入穿梭载体中以产生五个微型双元载体。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。将上述5个微型双元载体导入农杆菌中，通过组织培养法转化水稻，并通过浸花法（不进行组织培养）转化拟南芥。转化频率高，足以从水稻和拟南芥中获得许多转基因植株。Southern杂交分析表明，转基因水稻植株中整合了相对低拷贝数（1 ~ 3个）的T-DNA，尤其是在携带repA-LC + staA的pSTARH 301 G中。将该片段（mALS）与1.37或0.56 kb启动子区一起沿着亚克隆。将两个mALS盒分别引入两个微型双元载体pSTARH 301 G和pPARH 501 G中，以产生携带长或短mALS除草剂耐受标记基因的四个不同载体。通过农杆菌介导法将这4种载体导入水稻，均获得了较好的转化率，表明短启动子的mALS在水稻遗传转化中也能发挥良好的作用。