利用GUS基因的瞬时表达优化大豆根癌农杆菌介导的遗传转化
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摘要
高转化效率是分子育种的重要前提,根癌农杆菌介导的遗传转化由于其有较高的遗传效率和较低的拷贝数成为目前优选方法。为优化大豆遗传转化体系,以本实验室已有转化体系为基础,利用pCAMBIA3301中35S∶GUS基因瞬时表达体系,从萌发时间、切豆方法、侵染时间、共培养方式以及共培养时间等方面对东农47、垦农18和B12088大豆栽培品种的遗传转化体系进行优化。结果表明:东农47萌发1 d,侵染30 min;垦农18和B12088萌发12 h,侵染30 min,GUS基因瞬时表达率最高。在其它的处理上,3个品种表现结果一致,即蘸取菌液切豆,共培养中子叶伤口朝下摆放,黑暗条件下共培养3～5 d,GUS基因瞬时表达最高。3个品种在最适条件下,GUS染色效率均达到90%以上,垦农18遗传转化效率较高。本研究为大豆农杆菌介导子叶节转化方法提供了一个改进的方案,并且为拓宽可利用于大豆遗传转化的种质资源奠定基础。
High transformation efficiency is an important factor for plant molecular breeding. Agrobacterium tumefaciens-mediated transformation is commonly used in plant genetic transformation because its higher heritability and lower copy number. To optimize the soybean genetic transformation system used in our laboratory, we optimized Agrobacterium tumefaciens-mediated transformations of Dongnong 47, Kennong 18 and B12088 soybean varieties of germination time, bean cutting methods, infestation time, co-culture method and co-culture time with the expression of 35 S∶GUS of pCAMBIA3301. The results showed that the expression level of GUS was highest when Dongnong 47 was germinated for 1 d, infested for 30 min and Kennong 18 and B12088 germinated for 12 h, infected for 30 min. In other treatments, the results of the three varieties were consistent. The GUS gene expression was highest when we cut the soybean cotyledonary with dipped Agrobacterium and when the cotyledon′s wound is placed face down and co-cultivated for 3-5 d in the dark. Under the optimal conditions, the Kennong 18 showed the highest transformation efficiency. This study provides an improved protocol for Agrobacterium tumefaciens-mediated transformation of soybean cotyledonary nodes that can be utilized for soybean genetic transformation.
High transformation efficiency is an important factor for plant molecular breeding. Agrobacterium tumefaciens-mediated transformation is commonly used in plant genetic transformation because its higher heritability and lower copy number. To optimize the soybean genetic transformation system used in our laboratory, we optimized Agrobacterium tumefaciens-mediated transformations of Dongnong 47, Kennong 18 and B12088 soybean varieties of germination time, bean cutting methods, infestation time, co-culture method and co-culture time with the expression of 35 S∶GUS of pCAMBIA3301. The results showed that the expression level of GUS was highest when Dongnong 47 was germinated for 1 d, infested for 30 min and Kennong 18 and B12088 germinated for 12 h, infected for 30 min. In other treatments, the results of the three varieties were consistent. The GUS gene expression was highest when we cut the soybean cotyledonary with dipped Agrobacterium and when the cotyledon′s wound is placed face down and co-cultivated for 3-5 d in the dark. Under the optimal conditions, the Kennong 18 showed the highest transformation efficiency. This study provides an improved protocol for Agrobacterium tumefaciens-mediated transformation of soybean cotyledonary nodes that can be utilized for soybean genetic transformation.
引文
[1] 王志坤,李文滨.转基因科普系列—转基因大豆[J].大豆科技,2017(2):52-53.(Wang Z K,Li W B.Genetically modified popular science series-genetically modified soybeans[J].Soybean Technology,2017(2):52-53.)
[2] 常健敏,李丹丹,Arun S,等.转SiDGAT1高油大豆T-3株系的鉴定[J].作物杂志,2014(5):219-259.(Chang J M,Li D D,Arun S,et al.Identification of transgenic SiDGAT1 high oil soybean T-3 strain[J].Crops,2014(5):219-259.)
[3] Yang X.Analysis of the copy number of exogenous genes in transgenic cotton using real-time quantitative PCR and the 2-ΔΔCT method[J].African Journal of Biotechnology,2012,11(23),6226-6233.
[4] Somers D A,Samac D A,Olhoft P M.Recent advances in legume transformation[J].Plant Physiology,2003,131:892-899.
[5] Hinchee M A,Connor Ward D V,Newell C A,et al.Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer[J].Biotechnology,1988,6(8):915-922.
[6] Olhoft P M,Flagel L E,Donovan C M.Efficient soybean transfonnation using hygromycin B selection in the cotyledonary-node method[J].Planta,2003,216(5):723-735.
[7] Olthoft P M,Somer D A.L-cysteine increases Agrobacterium mediated T-DNA delivery into soybean cotyledonary node cells[J].Plant Cell Reports,2001,20(8):706-711.
[8] Xue R G,Xie H F,Zhang B.A multi-needle-assisted transforma-tion of soybean cotyledonary node cells[J].Biotechnology Letters,2006,28(19):1551-1557.
[9] 陈李淼,田星星,单志慧,等.利用农杆菌介导法转化大豆子叶节的影响因素研究[J].大豆科学,2012,31(1):17-23.(Chen L M,Tian X X,Shan Z H,et al.Optimization of the factors affecting genetic transformation of soybean coty-ledonary node mediated by agrobacterium tumefacines[J].Soybean Science,2012,31(1):17-23.)
[10] 翟锐,高乐,丁雪妮,等.农杆菌介导大豆子叶节遗传转化体系的优化[J].大豆科学,2015,34(5):768-775.(Zhai R,Gao L,Ding X N,et al.Optimization of cotyledonary-node agrobacterium-mediated soybean transformation system[J].Soybean Science,2015,34(5):768-775.)
[11] Dang W,Wei Z M.An optimized agrobacterium-mediated transformation for soybean for expression of binary insect resistance genes[J].Plant Science,2007,173(4):381-389.
[12] Li S X,Cong Y H,Liu Y P,et al.Optimization of agrobacterium-mediated transformation in soybean[J].Frontiers in Plant Science,2017,8:246.
[13] Jefferson R A,Kavanagh T A,Bevan M W.GUS fusions:β-glucuronidase as a sensitive and versatile gene fusion marker in higherplants[J].The EMBO Journal,1987,6(13):3901-3907.
[14] 余永亮,梁慧珍,王树峰,等.中国转基因大豆的研究进展及其产业化[J].大豆科学,2010,29(1):143-150.(Yu Y L,Liang H Z,Wang S F,et al.Research progress and commercialization on transgenic soybean in China[J].Soybean Science,2010,29(1):143-150.)
[15] 应珊,何晓薇,王秀荣,等.影响农杆菌介导的大豆转化效率的因素研究[J].分子植物育种,2008,6(1):32-40.(Ying S,He X W,Wang X R,et al.Assessment of factors affecting the transformation efficiency of soybean cotyledonary-node agrobacterium-mediated transformation system [J].Molecular Plant Breeding,2008,6(1):32-40.)
[2] 常健敏,李丹丹,Arun S,等.转SiDGAT1高油大豆T-3株系的鉴定[J].作物杂志,2014(5):219-259.(Chang J M,Li D D,Arun S,et al.Identification of transgenic SiDGAT1 high oil soybean T-3 strain[J].Crops,2014(5):219-259.)
[3] Yang X.Analysis of the copy number of exogenous genes in transgenic cotton using real-time quantitative PCR and the 2-ΔΔCT method[J].African Journal of Biotechnology,2012,11(23),6226-6233.
[4] Somers D A,Samac D A,Olhoft P M.Recent advances in legume transformation[J].Plant Physiology,2003,131:892-899.
[5] Hinchee M A,Connor Ward D V,Newell C A,et al.Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer[J].Biotechnology,1988,6(8):915-922.
[6] Olhoft P M,Flagel L E,Donovan C M.Efficient soybean transfonnation using hygromycin B selection in the cotyledonary-node method[J].Planta,2003,216(5):723-735.
[7] Olthoft P M,Somer D A.L-cysteine increases Agrobacterium mediated T-DNA delivery into soybean cotyledonary node cells[J].Plant Cell Reports,2001,20(8):706-711.
[8] Xue R G,Xie H F,Zhang B.A multi-needle-assisted transforma-tion of soybean cotyledonary node cells[J].Biotechnology Letters,2006,28(19):1551-1557.
[9] 陈李淼,田星星,单志慧,等.利用农杆菌介导法转化大豆子叶节的影响因素研究[J].大豆科学,2012,31(1):17-23.(Chen L M,Tian X X,Shan Z H,et al.Optimization of the factors affecting genetic transformation of soybean coty-ledonary node mediated by agrobacterium tumefacines[J].Soybean Science,2012,31(1):17-23.)
[10] 翟锐,高乐,丁雪妮,等.农杆菌介导大豆子叶节遗传转化体系的优化[J].大豆科学,2015,34(5):768-775.(Zhai R,Gao L,Ding X N,et al.Optimization of cotyledonary-node agrobacterium-mediated soybean transformation system[J].Soybean Science,2015,34(5):768-775.)
[11] Dang W,Wei Z M.An optimized agrobacterium-mediated transformation for soybean for expression of binary insect resistance genes[J].Plant Science,2007,173(4):381-389.
[12] Li S X,Cong Y H,Liu Y P,et al.Optimization of agrobacterium-mediated transformation in soybean[J].Frontiers in Plant Science,2017,8:246.
[13] Jefferson R A,Kavanagh T A,Bevan M W.GUS fusions:β-glucuronidase as a sensitive and versatile gene fusion marker in higherplants[J].The EMBO Journal,1987,6(13):3901-3907.
[14] 余永亮,梁慧珍,王树峰,等.中国转基因大豆的研究进展及其产业化[J].大豆科学,2010,29(1):143-150.(Yu Y L,Liang H Z,Wang S F,et al.Research progress and commercialization on transgenic soybean in China[J].Soybean Science,2010,29(1):143-150.)
[15] 应珊,何晓薇,王秀荣,等.影响农杆菌介导的大豆转化效率的因素研究[J].分子植物育种,2008,6(1):32-40.(Ying S,He X W,Wang X R,et al.Assessment of factors affecting the transformation efficiency of soybean cotyledonary-node agrobacterium-mediated transformation system [J].Molecular Plant Breeding,2008,6(1):32-40.)
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