【作者】 张家银；

【导师】 邓子牛；

【作者基本信息】 湖南农业大学 ， 果树学， 2008， 硕士

【摘要】 柑橘在世界农业经济中占有重要地位,但其产业发展一直受到柑橘各种病害的制约。栽培措施与农药对柑橘病害的防治能力有限,且化学农药还会造成严重的环境污染,抗病育种成为解决柑橘病害的必然选择。柑橘常规育种受到育种周期长、珠心胚干扰、性器官败育、遗传上高度杂合等因素影响而进程缓慢,同时物种间生殖隔离亦阻碍了常规育种进程。转基因技术的应用与抗病相关基因的克隆为柑橘抗病育种提供了新的思路。本研究采用农杆菌介导法,以柑橘溃疡病致病基因pthA的核定位信号(NLS)和抗真菌病害基因chit42为外源目的基因,分别将其转入到糖橙和椪柑中,通过嫁接使抗性芽再生成苗。采用PCR和RT-PCR等技术对转基因材料进行鉴定分析;通过离体接种病菌实验对转基因植株的抗病能力进行评价。主要研究结果如下:1.构建了PBI121-NLS植物表达载体;用农杆菌介导法将pthA-NLS基因导入到糖橙中,并经PCR,RT-PCR和Southern blot杂交分析获得9个转基因株系;转基因植株叶片离体接种柑橘溃疡病菌,结果表明转基因植株对柑橘溃疡病菌有很强的抗性。2.建立了椪柑实生苗上胚轴节间茎段的离体再生体系,结果表明:茎段在MS基本培养基中的萌芽率最高,可以达到83.3%,且萌芽时间短,多为单芽,芽体健壮,嫁接成活率高;抗生素筛选试验表明卡拉霉素(Km)100mg/l时可以有效控制逃逸体和嵌合体的产生;将抗性芽通过嫁接成苗,取新叶提取DNA,通过PCR初步鉴定,18株抗性植株中有3株为转基因植株。3.采用了7种消毒方法对椪柑成年态节间茎段消毒,结果表明“改良消毒法”可有效降低其污染率和死亡率,分别可降至12.2%和27.2%;椪柑成年态节间茎段在MS+1mg/l BA+0.5 mg/l NAA中的不定芽萌芽率最高,达12.8%:选用5 mm以下的茎尖可明显提高嫁接成活率,不过,使用1 mm左右茎尖进行微芽嫁接嫁接成活率最高,这可能与柑橘接穗越大携带的内生菌越多有关。通过农杆菌介导法将目的基因chit42转入到椪柑中,获得12棵抗性芽,PCR初步检测表明获得1棵转基因植株。更多还原

【Abstract】 Citrus is an important fruit crop for agricultural economy in the world, and the production is inhibited by citrus disease which severely occurres worldwide. Agronomic practice and bactericide can only solve the problem in some context, and some bactericide can cause heavy environmental pollution. These make the disease resistance breeding a best selection for fighting against the bacterial pathogens. Conventional disease resistance breeding is difficult because of polyembryony, apomixis, long juvenility, high heterozygosity and parthenogenesis; as well as limitation for reproduction barrier among different species. Resistant genes cloning and transgenic approach offers a potentially bactericide-free and environment-friendly solution for disease pathogen control. In this study, pthA-NLS gene and Chit42 gene were introduced into Citrus sinensis (Linn.) Osbeck and Ponkan (Citrus reticulata Blanco) by Agrobacterium-medialed transformation. Shoot-tip grafting was performed to complete the regeneration system.PCR and RT-PCR were used to analyze the transgenic plants.Disease resistance test indicated that resistance was improved on some transgenic strains. And the resistance mechanism of pthA-NLS in citrus was discussed. The main results are as follows:1. The plasmid of ’pthA-NLS’ was constructed and transfered to Citrus sinensis (Linn.) Osbeck by Agrobacterium-mediated. Nine transgenic strains were obtained through PCR, RT-PCR and Southern blot analysis. Disease resistance test indicated that resistance to canker was obviously improved on some transgenic strains.2. An efficient regeneration system by direct organogenesis from epicotyl of Ponkan (Citrus reticulata Blanco) in vitro was set up. Seven methods were tested for regeneration, MS without any hormone resulted in the most effective one, with the rates of bud formation efficiency to 83.3% and much stronger buds were got in shortest time .100mg/l Km was used to control chimeric buds. Shoot-tip grafting was performed to complete the regeneration system. Three trangenic strains were confirmed from 18 strains through PCR analysis.3. Seven methods were tested for the sterilization of mature materials, and the combination of pre-sampling and post-sampling disinfection (innovation method) resulted in the most effective one, with the rates of the contamination and death reduced to 12.2% and 27.2%, respectively. After sterilization with the innovation method, the percentage of the alive internodal segments rates raised to 30.6%. Furthermore,the medium supplemented with 1 mg/l BA and 0.5 mg/l NAA was the best one. The regeneration frequency and bud formation efficiency reached to 12.8%. Shoot-tip grafting was performed to complete the regeneration system. It was found that the buds smaller than 5 mm in length gave higher graft success than that with longer shoots, and the best results were obtained with 1 mm buds. This probably due to more endo-fungi existed in the bigger shoots than in the shorter ones.One buds of 12 was confirmed to transfenic strain by PCR analysis.更多还原