根癌农杆菌介导转化鄂抗棉9号愈伤组织获得含Chitinase和β-（1，3）-glucanase基因的棉花再生植株
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摘要
棉花(Gossypium hirsutum)是世界性的重要经济作物，在我国栽培面积较大，年种植面积达400万公顷，为我国农村经济的发展做出了重要贡献。而棉花的生产受到多种病原真菌的危害，随着种植面积的增加，棉花真菌病害在我国主要棉区迅速蔓延，严重影响了棉花的生产，造成了巨大经济损失。由于难以找到棉花抗源，因而利用广谱性抗真菌基因被认为是一个被看好的研究策略。几丁质酶和β-(1，3)-葡聚糖是绝大多数病原真菌细胞壁的组成成分，几丁质酶和β-(1，3)-葡聚糖酶通过降解病原真菌细胞壁达到抑制病原真菌的目的。
本试验利用根癌农杆菌介导的转基因方法，将双价抗真菌基因几丁质酶基因(Chitinase)和β-(1，3)葡聚糖酶基因(Glucanase)导入鄂抗棉9号胚性愈伤组织，结果表明：
1．IBA和KT及无机盐对鄂抗棉9号下胚轴再生有重要影响。在1.0mg／l IBA和0.5mg／l KT时为最佳配比，在愈伤组织诱导和增殖期间，培养基中分别添加120mgl~(-1)的硫酸镁和42mgl~(-1)硫酸亚铁，使愈伤向胚分化方向发育。在诱导愈伤时NH_4NO_3是不可缺少的，否则体细胞难以分化再生，但向胚性愈伤转变时，NH_4~+必须撤去，同时补加1900mgl~(-1)的KNO_3，KNO_3能促使胚的分化；到胚分化生长成苗时，需附加谷氨酰胺和天门冬酰胺，分别为1000mgl~(-1)和500mgl~(-1)，可以增加胚分化率和利于胚生长。
2．优化了根癌农杆菌介导的棉花胚性愈伤组织转化体系。以鄂抗棉9号愈伤组织为外植体，对转化的几个重要参数，如预培养的有无，共培养温度，乙酰丁香酮的有无，卡那霉素的选择浓度进行了筛选。结果表明：预培养5-10天较没有预培养的愈伤表现出更强的转化效率；共培养温度以19℃为适宜温度；使用乙酰丁香酮和不使用乙酰丁香酮效果不明显；卡那霉素的选择浓度以100mg／l为宜。
3．获得了60株抗性小苗，随机选取20株做PCR检测，有14株检测结果为阳性，取6株PCR阳性植株进行Southern检测，结果表明外源基因已经整合到棉花基因组中，拷贝数由1到3个不等。
4．对海岛棉的再生进行了初步研究。实验表明：2，4-D 0.15mg／l／KT 0.5mg／l时为新海14心形胚的诱导的最佳组合。
Upland cotton (Gossypium hirsutum) is one of the most important world wide economical crops, playing a vital role in the economic, political, and social affair. It is widely planted in our country, about 400 million hectors per year, and made a significant contribution to the development of rural area .while cotton is highly susceptible to many biotic and abiotic stresses, with the enlargement of its planting area, the disease of epiphyte is become increasingly overwhelming, thus endangered the quantity and quality of cotton production, and caused great calamity to cotton industry of our country .for the difficulty of obtain resistance material. It is a better strategy of useing abroad antifungal genes for transformation. To the property of chitinase and (3-(l,3)-glucanase can degrade the wall of fungal cell, thus it can be used to suppress fungal disease .
In this study chitinase and p-(l,3)-glucanase was transferred into Gossypium hirsutum E-kang-9 variety callus by Agrobacterium tumefaciens .The main results as follows:
1. In this research the affect of IBA & KT and inorganic salts to the regeneration of hypocotyls was studied, the primary callus initiation on the medium containing the optimal combination of 1.0 mg/L IBA and 0.5 mg/L KT, When primary callus are induced and proliferated on the medium orderly supplemented with 120 mg/L MgSO4and 42 mg/L FeSO4, calli was prone to form embryogenic calli. 1000 mg/L KCl and 200 mg/l CaCl2, and the callus is quickly promoted to form embryogenic calli. During calli inducing NH4NO3 is inevitable otherwise somatic embryo will not formed ,but during the stage of somatic embryo transformed to calli, NIHUNO3 must deserted and supplied with 1900mg/lKNO3, for it can benefit to the form of calli; Asn and Gin should be added during embryos germinate to young plants.
2. In this study efficient callus transformation system mediated by Agrobacterium was developed using E-R 9 embryogenic callus as explants, optimized the parameter about transformation such as pre-cultivation, co-cultivation temperature, AS and Km concentration .The result as follow: The efficiency of transformation for 5 to 10 days pre-cultivation is super to not pre-cultivation ; the best choice temperature of co-cultivation is 19℃; AS is not necessary to this experiment; The suitable concentration of Km is 100mg/l.
3. In this study 60 resistant plantlets were obtained, random select 20 plantlets, PCR result verified 14 plantlets to be positive.
4.Preliminary study of embryogenic regeneration on Gossypium barbadense was investigated .The results show us: The combination of 2,4-D 0.15 mg/1 and KT 0.5 mg/1 is
the best one on calli inducing of Gossypium barbadens
本试验利用根癌农杆菌介导的转基因方法，将双价抗真菌基因几丁质酶基因(Chitinase)和β-(1，3)葡聚糖酶基因(Glucanase)导入鄂抗棉9号胚性愈伤组织，结果表明：
1．IBA和KT及无机盐对鄂抗棉9号下胚轴再生有重要影响。在1.0mg／l IBA和0.5mg／l KT时为最佳配比，在愈伤组织诱导和增殖期间，培养基中分别添加120mgl~(-1)的硫酸镁和42mgl~(-1)硫酸亚铁，使愈伤向胚分化方向发育。在诱导愈伤时NH_4NO_3是不可缺少的，否则体细胞难以分化再生，但向胚性愈伤转变时，NH_4~+必须撤去，同时补加1900mgl~(-1)的KNO_3，KNO_3能促使胚的分化；到胚分化生长成苗时，需附加谷氨酰胺和天门冬酰胺，分别为1000mgl~(-1)和500mgl~(-1)，可以增加胚分化率和利于胚生长。
2．优化了根癌农杆菌介导的棉花胚性愈伤组织转化体系。以鄂抗棉9号愈伤组织为外植体，对转化的几个重要参数，如预培养的有无，共培养温度，乙酰丁香酮的有无，卡那霉素的选择浓度进行了筛选。结果表明：预培养5-10天较没有预培养的愈伤表现出更强的转化效率；共培养温度以19℃为适宜温度；使用乙酰丁香酮和不使用乙酰丁香酮效果不明显；卡那霉素的选择浓度以100mg／l为宜。
3．获得了60株抗性小苗，随机选取20株做PCR检测，有14株检测结果为阳性，取6株PCR阳性植株进行Southern检测，结果表明外源基因已经整合到棉花基因组中，拷贝数由1到3个不等。
4．对海岛棉的再生进行了初步研究。实验表明：2，4-D 0.15mg／l／KT 0.5mg／l时为新海14心形胚的诱导的最佳组合。
Upland cotton (Gossypium hirsutum) is one of the most important world wide economical crops, playing a vital role in the economic, political, and social affair. It is widely planted in our country, about 400 million hectors per year, and made a significant contribution to the development of rural area .while cotton is highly susceptible to many biotic and abiotic stresses, with the enlargement of its planting area, the disease of epiphyte is become increasingly overwhelming, thus endangered the quantity and quality of cotton production, and caused great calamity to cotton industry of our country .for the difficulty of obtain resistance material. It is a better strategy of useing abroad antifungal genes for transformation. To the property of chitinase and (3-(l,3)-glucanase can degrade the wall of fungal cell, thus it can be used to suppress fungal disease .
In this study chitinase and p-(l,3)-glucanase was transferred into Gossypium hirsutum E-kang-9 variety callus by Agrobacterium tumefaciens .The main results as follows:
1. In this research the affect of IBA & KT and inorganic salts to the regeneration of hypocotyls was studied, the primary callus initiation on the medium containing the optimal combination of 1.0 mg/L IBA and 0.5 mg/L KT, When primary callus are induced and proliferated on the medium orderly supplemented with 120 mg/L MgSO4and 42 mg/L FeSO4, calli was prone to form embryogenic calli. 1000 mg/L KCl and 200 mg/l CaCl2, and the callus is quickly promoted to form embryogenic calli. During calli inducing NH4NO3 is inevitable otherwise somatic embryo will not formed ,but during the stage of somatic embryo transformed to calli, NIHUNO3 must deserted and supplied with 1900mg/lKNO3, for it can benefit to the form of calli; Asn and Gin should be added during embryos germinate to young plants.
2. In this study efficient callus transformation system mediated by Agrobacterium was developed using E-R 9 embryogenic callus as explants, optimized the parameter about transformation such as pre-cultivation, co-cultivation temperature, AS and Km concentration .The result as follow: The efficiency of transformation for 5 to 10 days pre-cultivation is super to not pre-cultivation ; the best choice temperature of co-cultivation is 19℃; AS is not necessary to this experiment; The suitable concentration of Km is 100mg/l.
3. In this study 60 resistant plantlets were obtained, random select 20 plantlets, PCR result verified 14 plantlets to be positive.
4.Preliminary study of embryogenic regeneration on Gossypium barbadense was investigated .The results show us: The combination of 2,4-D 0.15 mg/1 and KT 0.5 mg/1 is
the best one on calli inducing of Gossypium barbadens
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