本文關(guān)鍵詞：以草甘膦為篩選標(biāo)記的大豆轉(zhuǎn)基因體系的建立及抗除草劑轉(zhuǎn)基因大豆的培育，由筆耕文化傳播整理發(fā)布。

以草甘膦為篩選標(biāo)記的大豆轉(zhuǎn)基因體系的建立及抗除草劑轉(zhuǎn)基因大豆的培育

Establishment of an Efficient Transformation Protocol for Soybean Using Glyphosate As Selective Agent and the Development of Glyphosate-Tolerant Transgenic Soybean Lines

[1] [2] [3] [4] [5] [6] [7] [8]

LU LingHong1, HAN Qiang1, LI Lin1, ZHOU Lian1, LIU RuiFang1, SONG ZhangYue1, SHEN ZhiCheng2 ＆ SHOU HuiXiaI 1 State Key Laboratory of Plant Physiology and Biochemistry, Coll

[1]浙江大學(xué)生命科學(xué)學(xué)院植物生理學(xué)與生物化學(xué)國家重點(diǎn)實(shí)驗(yàn)室,杭州310058; [2]浙江大學(xué)農(nóng)業(yè)與生物技術(shù)學(xué)院昆蟲科學(xué)研究所,杭州310058

文章摘要：抗草甘膦轉(zhuǎn)基因大豆能顯著提高大豆生產(chǎn)效率，具有重大的應(yīng)用前景．本實(shí)驗(yàn)室前期研究建立了農(nóng)桿菌介導(dǎo)、草胺膦為篩選劑的大豆轉(zhuǎn)基因體系，轉(zhuǎn)化效率在4％以上．在此基礎(chǔ)上，利用G6．EPSPS和GIO—EPSPS2個(gè)具有自主知識(shí)產(chǎn)權(quán)的草甘膦抗性基因，通過優(yōu)化轉(zhuǎn)化體系，成功建立了以草甘膦為篩選劑的大豆遺傳轉(zhuǎn)化體系，轉(zhuǎn)化效率達(dá)1％以上．濃度梯度實(shí)驗(yàn)發(fā)現(xiàn)，當(dāng)草甘膦的篩選濃度為100mg／L時(shí)，雖然叢生芽的再生率下降了50％～60％，，但最終轉(zhuǎn)化效率不受影響．進(jìn)一步通過基因表達(dá)分析、Westernblot、Southernblot和除草劑抗性鑒定等方法對(duì)轉(zhuǎn)基因大豆進(jìn)行了分子檢測(cè)和驗(yàn)證，最后獲得了分子特征明確、對(duì)草甘膦抗性穩(wěn)定的抗草甘膦轉(zhuǎn)基因大豆后代．結(jié)果對(duì)國內(nèi)抗草甘膦轉(zhuǎn)基因大豆轉(zhuǎn)基因方法研究及抗除草劑新品種選育具有意義．

Abstr：Herbicide-tolerant soybean can significantly reduce the weed control cost, thus has great potential applications in agriculture. In a previous study, we described an efficient soybean transformation protocol using glufosinate as selective agent. The transformation efficiency was up to 4%. In this study, we attmpted to establish a transformation system using the newly isolated EPSPS genes, G6-EPSPS and GIO-EPSPS as the selective marker gene. By optimization of the factors affecting each step of the transformation process, we were able to achieve a stable transformation system with efficiency higher than 1%. While the high glyphosate concentration （100 mg/L） signficantly reduced the numbers of proliferated shootlets in the cotyledonary nodes of soybean explants, the final transformation efficiency was not affected. Molecular analyses using Southern blot, reverse transcription PCR （RT-PCR）, and Western blot verfied the insertions of the transgene in the transgenic soybean lines. The transgenic soybean plants were able to tolerant to spray of Roundup~ up to 6 kg/hectares, which is 4.9 folds of the recommended field application rate. These results are valuable for soybean trangenic research and development of transgenic soybean for field application.

文章關(guān)鍵詞：

Keyword：:soybean, transformation, glyphosate, molecular verification

課題項(xiàng)目：轉(zhuǎn)基因生物新品種培育科技重大專項(xiàng)（批準(zhǔn)號(hào)：2013zx08004.OOl,2014zx08004.01B）資助

作者信息：會(huì)員可見

  本文關(guān)鍵詞：以草甘膦為篩選標(biāo)記的大豆轉(zhuǎn)基因體系的建立及抗除草劑轉(zhuǎn)基因大豆的培育，由筆耕文化傳播整理發(fā)布。