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

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.