苎麻抗虫基因遗传转化研究
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摘要
苎麻（Boehmeria nivea L. Gand）为荨麻科苎麻属的多年生宿根性草本植物，主要分布在热带和亚热带地区，为我国特产。苎麻纤维是优良的精纺纤维。由于苎麻为高度杂合体，其遗传背景十分复杂，有性杂交等常规方法育种存在周期长、工作量大和效率低等问题，难以取得育种上的突破性进展。利用外源基因进行苎麻定向遗传改良、培育新的种质资源或品种是解决问题的有效途径。苎麻转基因研究虽有报道，但这些研究大多以证明根癌农杆菌介导的苎麻遗传转化方法的可行性为目的，并没有优化转化条件。
本研究从建立高频再生体系入手，建立了农杆菌介导、基因枪和农杆菌介导花粉侵入法等三种遗传转化方法，将双价抗虫基因导入到具有重要推广价值的栽培苎麻品种中。主要研究结果如下：
（1）建立苎麻下胚轴和子叶高频再生体系
在大量前期激素配比试验的基础上，研究了BA和TDZ激素配比对苎麻下胚轴直接再生的作用、TDZ与GA3和IAA配合使用对下胚轴愈伤组织诱导不定芽的影响和丛生芽再生所需的生长调节剂配比。基于以上研究，确立了TDZ的浓度为0.045μM、GA3为0.35μM和IAA为0.045μM作为农杆菌转化的再生体系激素组合。通过对中苎1号、中饲苎1号、NC01三个品种的下胚轴和子叶的再生试验，验证了该再生体系的适应性。
（2）建立农杆菌介导苎麻下胚轴和子叶遗传转化体系
在建立高频再生体系的基础上，对农杆菌介导苎麻下胚轴遗传转化的侵染时间、预培养时间、共培养时间、抗生素浓度、Kan筛选浓度再生和生根浓度等主要影响因素进行了系统研究，优化了转化流程中的各项参数，建立了农杆菌介导苎麻下胚轴遗传转化体系。对获得的抗性植株进行了PCR检测、RT-PCR、GUS组织染色和Southern杂交等检测。并通过三个基因型子叶和下胚轴转化试验，验证了该遗传转化体系的适应性。
（3）建立苎麻基因枪遗传转化体系
基于本实验室对茎尖再生体系的研究基础和第一章对子叶再生体系，对基因枪转化的轰击距离、不同金粉及DNA用量、轰击压力和轰击次数等主要影响因素进行了研究，确定了转化流程中的各项参数，对获得的抗性植株进行了PCR检测和GUS组织染色，建立了基因枪茎尖和子叶愈伤组织遗传转化体系。
（4）建立农杆菌介导苎麻花粉侵入法遗传转化体系
通过对表面活性剂浓度、侵染方式、侵染时间、侵染次数等主要影响因素进行了系统研究，确立了转化流程，建立了农杆菌介导苎麻花粉侵入法遗传转化体系。建立了T1代抗性种子的筛选体系，对获得的抗性植株进行了PCR检测、荧光定量PCR、GUS组织染色和Southern杂交等检测。
Ramie( Boehmeria nivea Gaud)is a perennial herbaceous plant of the Urticaceae family. Ramie bast fiber is a kind of fine plant texile materials. It is mainly planted in tropical and subtropical regions in China.It is difficult to achieve a breakthrough in breeding duo to its high heterozygotes, complex genetic background, long sexual hybridization breeding cycle. So development of a genetic transformation system to introduce desired genes into ramie could improve quality, develop new germplasm or cultivars. Several studies have successfully developed transgenic ramie by using A. tumefaciens-mediated transformation, but in most cases the transformation efficiency was low. All these studies emphasized on the feasibility of Agrobacterium-mediated transformation to generate transgenic ramie and the protocol has not been optimized.
In this study, an efficient regeneration system for adventitious shoot induction was developed by using hypocotyls explants of ramie, either by shoot organogenes or multiple shoot formation from callus, and by direct regeneration of shoots without any intermediate callus phase. Transformation system of ramie was established via Agrobacterium tumefaciens-mediated method , particle bombardment and the floral dip method, transgenic plants integrated with an insect-resistance gene were produced.Main research results as follows:
(1)The establishment of efficient protocols for plant regeneration from hypocotyls and cotyledon
Based on pre-trial, we compared the responses of ramie hypocotyls segments on various TDZ concentrations in combination with different concentrations of BA to effect of direct shoot regeneration without an intermediate callus phase,a two-step procedure for efficient indirect regeneration by and multiple shoot formation. Based on the above study, two-step procedure for efficient indirect regeneration was chosen for the transformation system of ramie via Agrobacterium tumefaciens-mediated method due to its highest efficiency of shoot formation. Cotyledon and hypocotyl explants of var. Zhongzhu No.1 Zhongsizhu No.1 NC01was tested for feasibility of this regeneration protocol.
(2)The establishment of Genetic transformation of ramie by Agrobacteriumtumefaciens-mediated method from hypocotyls and cotyledon
Based on the above efficient protocols for plant regeneration, the effect of infection time, selection pressure , co-cultivation time, pre-cultivation time and concentration of cefotaxime on transient GUS expression and regeneration efficiency were evaluated and an efficient transformation system by Agrobacteriumtumefaciens-mediated was established. Cotyledon and hypocotyl explants of var. Zhongzhu No.1 Zhongsizhu No.1 NC01was tested for feasibility of this transformation protocol. GUS detection, PCR detection, RT-PCR and Southern blotting detection were used to detect putative transformed plants, while showed that foreign gene had been integrated into ramie genome.
(3)The establishment of genetic transformation of ramie by particle bombardment
Based on research shoot-tip regeneration by our lab and the above efficient protocols for cotyledon regeneration, the effect of helium pressure, the vaccum pressure, target distance and times of bombardment.on transient GUS expression and were evaluated and an efficient transformation system by particle bombardment was established. Putative transformed plants were detected by GUS and PCR.
(4)The establishment of genetic transformation of ramie by floral dip method
The effect of concentration of Silwet L-77, infection of time, times of infection, drop-by-drop inoculation and submersion on the efficiency transformation and were evaluated and an efficient transformation system by floral dip method was established. T1 transformed plants was determined experimentally, by planting wild type plants on MS medium supplemented with a range of concentrations of the kanamycin. GUS detection, PCR detection, fluorescence quantitative PCR and Southern blotting detection were used to detect putative transformed plants, while showed that foreign gene had been integrated into ramie genome.
本研究从建立高频再生体系入手，建立了农杆菌介导、基因枪和农杆菌介导花粉侵入法等三种遗传转化方法，将双价抗虫基因导入到具有重要推广价值的栽培苎麻品种中。主要研究结果如下：
（1）建立苎麻下胚轴和子叶高频再生体系
在大量前期激素配比试验的基础上，研究了BA和TDZ激素配比对苎麻下胚轴直接再生的作用、TDZ与GA3和IAA配合使用对下胚轴愈伤组织诱导不定芽的影响和丛生芽再生所需的生长调节剂配比。基于以上研究，确立了TDZ的浓度为0.045μM、GA3为0.35μM和IAA为0.045μM作为农杆菌转化的再生体系激素组合。通过对中苎1号、中饲苎1号、NC01三个品种的下胚轴和子叶的再生试验，验证了该再生体系的适应性。
（2）建立农杆菌介导苎麻下胚轴和子叶遗传转化体系
在建立高频再生体系的基础上，对农杆菌介导苎麻下胚轴遗传转化的侵染时间、预培养时间、共培养时间、抗生素浓度、Kan筛选浓度再生和生根浓度等主要影响因素进行了系统研究，优化了转化流程中的各项参数，建立了农杆菌介导苎麻下胚轴遗传转化体系。对获得的抗性植株进行了PCR检测、RT-PCR、GUS组织染色和Southern杂交等检测。并通过三个基因型子叶和下胚轴转化试验，验证了该遗传转化体系的适应性。
（3）建立苎麻基因枪遗传转化体系
基于本实验室对茎尖再生体系的研究基础和第一章对子叶再生体系，对基因枪转化的轰击距离、不同金粉及DNA用量、轰击压力和轰击次数等主要影响因素进行了研究，确定了转化流程中的各项参数，对获得的抗性植株进行了PCR检测和GUS组织染色，建立了基因枪茎尖和子叶愈伤组织遗传转化体系。
（4）建立农杆菌介导苎麻花粉侵入法遗传转化体系
通过对表面活性剂浓度、侵染方式、侵染时间、侵染次数等主要影响因素进行了系统研究，确立了转化流程，建立了农杆菌介导苎麻花粉侵入法遗传转化体系。建立了T1代抗性种子的筛选体系，对获得的抗性植株进行了PCR检测、荧光定量PCR、GUS组织染色和Southern杂交等检测。
Ramie( Boehmeria nivea Gaud)is a perennial herbaceous plant of the Urticaceae family. Ramie bast fiber is a kind of fine plant texile materials. It is mainly planted in tropical and subtropical regions in China.It is difficult to achieve a breakthrough in breeding duo to its high heterozygotes, complex genetic background, long sexual hybridization breeding cycle. So development of a genetic transformation system to introduce desired genes into ramie could improve quality, develop new germplasm or cultivars. Several studies have successfully developed transgenic ramie by using A. tumefaciens-mediated transformation, but in most cases the transformation efficiency was low. All these studies emphasized on the feasibility of Agrobacterium-mediated transformation to generate transgenic ramie and the protocol has not been optimized.
In this study, an efficient regeneration system for adventitious shoot induction was developed by using hypocotyls explants of ramie, either by shoot organogenes or multiple shoot formation from callus, and by direct regeneration of shoots without any intermediate callus phase. Transformation system of ramie was established via Agrobacterium tumefaciens-mediated method , particle bombardment and the floral dip method, transgenic plants integrated with an insect-resistance gene were produced.Main research results as follows:
(1)The establishment of efficient protocols for plant regeneration from hypocotyls and cotyledon
Based on pre-trial, we compared the responses of ramie hypocotyls segments on various TDZ concentrations in combination with different concentrations of BA to effect of direct shoot regeneration without an intermediate callus phase,a two-step procedure for efficient indirect regeneration by and multiple shoot formation. Based on the above study, two-step procedure for efficient indirect regeneration was chosen for the transformation system of ramie via Agrobacterium tumefaciens-mediated method due to its highest efficiency of shoot formation. Cotyledon and hypocotyl explants of var. Zhongzhu No.1 Zhongsizhu No.1 NC01was tested for feasibility of this regeneration protocol.
(2)The establishment of Genetic transformation of ramie by Agrobacteriumtumefaciens-mediated method from hypocotyls and cotyledon
Based on the above efficient protocols for plant regeneration, the effect of infection time, selection pressure , co-cultivation time, pre-cultivation time and concentration of cefotaxime on transient GUS expression and regeneration efficiency were evaluated and an efficient transformation system by Agrobacteriumtumefaciens-mediated was established. Cotyledon and hypocotyl explants of var. Zhongzhu No.1 Zhongsizhu No.1 NC01was tested for feasibility of this transformation protocol. GUS detection, PCR detection, RT-PCR and Southern blotting detection were used to detect putative transformed plants, while showed that foreign gene had been integrated into ramie genome.
(3)The establishment of genetic transformation of ramie by particle bombardment
Based on research shoot-tip regeneration by our lab and the above efficient protocols for cotyledon regeneration, the effect of helium pressure, the vaccum pressure, target distance and times of bombardment.on transient GUS expression and were evaluated and an efficient transformation system by particle bombardment was established. Putative transformed plants were detected by GUS and PCR.
(4)The establishment of genetic transformation of ramie by floral dip method
The effect of concentration of Silwet L-77, infection of time, times of infection, drop-by-drop inoculation and submersion on the efficiency transformation and were evaluated and an efficient transformation system by floral dip method was established. T1 transformed plants was determined experimentally, by planting wild type plants on MS medium supplemented with a range of concentrations of the kanamycin. GUS detection, PCR detection, fluorescence quantitative PCR and Southern blotting detection were used to detect putative transformed plants, while showed that foreign gene had been integrated into ramie genome.
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