农杆菌介导Chi-linker-Glu融合基因和bar基因转化玉米茎尖的研究

草业学报 ›› 2012, Vol. 21 ›› Issue (5): 69-76.

农杆菌介导Chi-linker-Glu融合基因和bar基因转化玉米茎尖的研究

方永丰^1,2,3,李永生²,彭云玲³,王芳³,王威³,穆延召²,王汉宁^1,2,3*

1.甘肃省作物遗传改良与种质创新重点实验室,甘肃兰州730070;
2.甘肃省干旱生境作物学重点实验室,甘肃兰州730070;
3.甘肃农业大学农学院,甘肃兰州 730070

收稿日期:2012-02-22 出版日期:2012-05-25 发布日期:2012-10-20
通讯作者: E-mail:wanghn@ gsau.edu.cn
作者简介:方永丰(1976-),男,甘肃临洮人,在读博士。E-mail:fangyf@ gsau.edu.cn
基金资助:
973计划前期研究专项(2012CB722902)和科技部农业科技成果转化资金项目(2010GB2G100484)资助。

Agrobacterium-mediated transformation of maize shoot apical meristem by introducing fused gene Chi-linker-Glu and bar

FANG Yong-feng^1,2,3, LI Yong-sheng², PEN Yun-ling², WANG Fang², WANG Wei³, MU Yan-zhao², WANG Han-ning^1,2,3

1.Gansu Key Laboratory of Crop Improvement and & Germplasm Enhancement, Lanzhou 730070, China;
2.Gansu Provincial Key Laboratory of Arid-land Crop Science, Lanzhou 730070, China;
3.College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China

Received:2012-02-22 Online:2012-05-25 Published:2012-10-20

摘要/Abstract

摘要： 真菌性病害和田间杂草严重影响着玉米产量及饲用品质,几丁质酶和β-1,3葡聚糖酶催化病原真菌细胞壁的水解反应,二者协同作用能够有效地抑制病原真菌生长。为了获得兼具真菌病及除草剂抗性的玉米种质新材料,本研究通过对农杆菌介导的玉米茎尖遗传转化体系的优化,将几丁质酶、β-1,3葡聚糖酶的融合基因以及bar基因导入优良玉米自交系郑58,用200 mg/L的Basta除草剂进行筛选,并对抗性植株进行了PCR检测。结果表明, LBA4404菌液浓度(OD₆₀₀值)为0.6、乙酰丁香酮浓度为150 μmol/L、50 kPa负压侵染12 min为最佳的遗传转化条件;对除草剂筛选后的32株抗性植株进行PCR鉴定,有13株为阳性植株,初步鉴定结果表明目的基因已经整合进玉米基因组中。

Abstract: Most maize disease are caused by fungi pathogens, such as northern leaf blight, southern leaf blight, ear and stalk rots, and so on. In normal years, this disease makes maize yield lost by 10% and this percentage may be increased by 30%-40% in its popular years. However, it is very difficult for conventional breeding to develop an elite maize material with the resistance to all the fungi disease. Therefore, we can use transgenic method to transfer some genes related to the resistance of these disease into maize in order to obtain resistance materials. Chitinase and β-1,3-glucanase are two important enzymes which play a key role in the hydrolysis reaction of fungal cell wall, so it is useful in the control of fungi disease, meanwhile, the herbicide resistant gene bar are usually used as a selectable marker in maize transformation.The purpose of this research is to introduce the fused gene of chitinase and β-1,3-glucanase and bar as well into elite maize inbred Zheng58 for improving its resistance to fungi disease. The wounded shoot apical meristem (SAM) of germinated seedlings were used as the material for Agrobacterium mediated transformation. We report here: 1) An optimized transformation system for Agrobacterium mediated transformation of shoot apical meristem, using the optimal concentration of bacterial culture (the value of OD₆₀₀ was 0.6) for infection, addition of 150 μmol/L acetosyringone (AS) in the bacterial suspension, the whole infection process was carried out in a vacuum desiccators with a negative pressure of 50 kPa for 12 minutes. 2) Identified by herbicide screening procedure and PCR detection, 13 transformed plants were obtained among the 32 herbicide-resistant plants, and the overall transformation was 2.6%.The preliminary evidences showed that the foreign genes had been introduced into the maize genome. This method circumvented the long period of tissue culture step and limitation of different seasons, in addition, many elite inbred lines which are recalcitrant in callus induction can be efficiently transformed by this method.

中图分类号:

方永丰,李永生,彭云玲,王芳,王威,穆延召,王汉宁. 农杆菌介导Chi-linker-Glu融合基因和bar基因转化玉米茎尖的研究[J]. 草业学报, 2012, 21(5): 69-76.

FANG Yong-feng, LI Yong-sheng, PEN Yun-ling, WANG Fang, WANG Wei, MU Yan-zhao, WANG Han-ning. Agrobacterium-mediated transformation of maize shoot apical meristem by introducing fused gene Chi-linker-Glu and bar[J]. Acta Prataculturae Sinica, 2012, 21(5): 69-76.

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