GmAOC3基因转化载体构建及转化大豆的初步研究

Construction of Transformation Vector of GmAOC3 Gene and Preliminary Study on the Transformation of Soybean

选用内切酶SacⅠ和MluⅠ切割目标基因和载体,构建了携带bar基因的重组载体p BA002-Gm AOC3,选用2个大豆品种(Jack和南农88-1)和2种外植体(子叶节和整个子叶节),研究大豆品种和外植体对根癌农杆菌介导的子叶节转化Gm AOC3基因的影响。结果表明:外植体为整个子叶节的大豆品种Jack的出芽率和转化率最高,分别为79.5%和2.27%。经Quick Stix PAT/bar基因试纸条检测、目标基因的分子检测和草丁膦抗性鉴定,共得到转Gm AOC3基因的T0代阳性株系3株,T1代阳性转基因大豆6株,其中5株以Jack品种为受体的转基因大豆Gm AOC3基因的表达量均显著高于非转基因植株,荧光定量拷贝数检测结果显示,4株转基因单株为单拷贝,2株为双拷贝。

Allene oxide cyclase（ AOC） plays a key role in the synthesis of jasmonic acid,which is an important defense signaling molecular in plant. In this study,we constructed a recombinant vector p BA002-Gm AOC3 with bar gene using double enzymes cleavage method（ Sac Ⅰ and Mlu Ⅰ） and the recombinant vector was successfully transformed into soybean via Agrobacterium transformation system. Two soybean varieties（ Jack and Nannong 88-1） and explants（ cotyledonary node and whole cotyledonary node） were used to study their effects on the Agrobacterium-mediated transformation of Gm AOC3. The results showed that the whole cotyledonary node of Jack had the highest budding rate（ 79. 5%） and conversion rate（ 2. 27%）.Based on the results of Quick Stix PAT / bar test paper detection,molecular detection of target DNA and Basta resistance test of leaves,we obtained three T0-generation Gm AOC3 positive transgenic soybean plants and six T1-generation Gm AOC3 positive transgenic soybean plants,of which five T1-generation transgenic plants from Jack showed higher expression level of Gm AOC3 than Jack. We also used the fluorescence quantitative PCR to detect the copy number of target gene. Besides two T1-generation transgenic plants with two copies of bar genes,the others contained one copy of bar gene. The data in this study would give some references to soybean genetic transformation and the Gm AOC3 transgenic plant obtained in this study could be used in soybean insect resistance breeding in future.