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

1.通过将复制蛋白repA[3种类型：高拷贝型、野生型和低拷贝型（LC）]与在农杆菌上具有质粒稳定性的staA或par片段适当结合，构建了能够在大肠杆菌和农杆菌中进行复制的非常小的质粒骨架。然后，将Pnos::Hyg^R标记和P35S::gusA报告带引入穿梭载体中，生成5个迷你二进制载体。该载体体积小（8.6 ~ 9.65 kb），携带Hyg^R和gusA基因间的4个多克隆位点。此外，通过用Gateway卡带替换gusA，我得到了三个Gateway型二进制向量。将上述5种微型二元载体导入农杆菌后，分别用组织培养法转化水稻和用花浸法（无组织培养）转化拟南芥。转化频率高到足以从水稻和拟南芥中获得许多转基因植株。Southern blot分析表明，相对较低拷贝数（1 - 3）的t - dna被整合到转基因水稻植株中，特别是当pSTARH301G携带repA-LC + staA.3进行转化时。对编码序列（CDS）中具有两个不同点突变的水稻乙酰乳酸合成酶（ALS）基因进行了进一步修改，删除了CDS中的单个HindIII位点。该片段（mALS）沿1.37或0.56 kb的启动子区域进行亚克隆。将2个mALS基因盒分别导入pSTARH301G和pPARH5O1G两个迷你二元载体中，得到4个携带mALS长、短抗除草剂标记基因的载体。这4种载体均通过农杆菌导入水稻，转化频率较高，表明即使启动子短的mALS也能很好地转化水稻。

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.