【作者】 马伟；

【导师】 崔崇士；

【作者基本信息】 东北农业大学 ， 作物遗传育种， 2002， 博士

【摘要】 大白菜是人们喜爱的大众蔬菜。而以芜菁花叶病毒（TuMV）为主的大白菜病害一直没有得到很好解决。蔬菜遗传转化可以利用的目的基因很多，但在大白菜转基因方面研究工作却很少。利用农杆菌介导法转化大白菜抗病基因的研究工作在国内外未见报道。本课试验采用农杆菌介导法将TuMV-CP基因导入大白菜中，建立了高效的大白菜离体再生、遗传转化体系，并对转基因植株进行分子生物学检测，证实得到的再生植株为转基因植株，目的基因已在部分植株上表达。同时，对转基因植株的后代进行检测，分析该基因所控制性状的遗传稳定性以及基因表达情况，为大白菜基因工程抗病育种提供理论依据。 试验得到结论如下： 1．建立了高效的大白菜离体再生体系 5日龄的大白菜二牛心品种的子叶，置于诱导芽分化的培养基（MS+5mg/L6-BA+0.5mg／L NAA+5mg/L AgNO₃）上，诱导不定芽数最多。置于生根培养基（1／2MS+0.2mg/L IBA），生根效果好，得到的再生植株多。 2．建立了大白菜遗传转化体系。 a．确立了大白菜转化过程中筛选剂及抑菌剂的种类及浓度。筛选剂为卡那霉素，最适筛选浓度为10mg／L；头孢曲松那的抑菌效果最好，最适浓度为300～500mg／L。 b．确立了农杆菌介导转化过程中基因型是影响转化率的主要因素。通过极差分析，基因型为“二牛心”，菌液浓度15倍的，外植体为子叶，感染方法为浸泡法是最佳的组合。农杆菌与外植体共培养的时间为28h。 c．试验表明所用的四个大白菜品种，品种“二牛心”子叶再生频率较高，品种“东农901”下胚轴再生频率较高。 d．比较了R1000与EHA105介导转化对抗性芽的影响：发根杆菌在浸染外植体时得到的抗性芽较多，得到的转基因植株相对少。而根癌农杆菌浸染外植体时得到抗性芽相 马 伟 摘 要2002年5月对少，得到的转基因植株相对多。 3．获得了大白菜抗病转基因植株。 大白菜转基因植株TO代，经PCR、PCR－Southern检测己证实导入目的基因己整合到植物基因组中，得到转基因植株15株己完全整合，但经RTNR检测，只有12株表达，证明一部分植株中有基因沉默现象。大白菜转基因植株乃代，经PCR、PCRSouthl检测，后代有分离现象，分离比例趋于3：l，符合盂德尔分离规律。 4．接病毒实验 经病毒接种试验表明：转基因＊代植株接种hlMv病毒后，发病率和病情指数明显低于对照品种。更多还原

【Abstract】 The Chinese cabbage is a kind of popular vegetable.But the Chinese cabbage disease hasn’t been solved till now. introduced infected by the turnip mosaic virus.There are many useful objective genes can be used in vegetable genetic transformation,but the research work of Chinese cabbage genetic transformation is little.The reaserch on transforming Chinese cabbage using Agrobacterium-med\ated method hasn’t been report at home and abroad.ln the test ,TuMV-CP gene was transferred into Chinese cabbage(Brassica campestris L.ssp.pekinensis) via Agrobacterium -mediated method.A high effective regeneration and genetic transformatin system has been established,The detection by the method of molecular biology, has proved that the regenerative plants are transgenic plants and the target genes have been expressed transgenic plants partly.Meanwhile transgenic progenies were traced and investigated so that heredity,stability and expression of target gene were researched.the virus resistant,stable plants were expected to obtain so that theoretical base can be established for Chinese cabbage breeding by gene engineering.Conclusion of this experiment are as following:1. A high effective regeneration system has been established.When five-day cotyledon of ErNiuXin cabbage,was placed on the polarization medium(MS+5mg/L6-BA+0.5mg/LNAA+5mg/LAgNO3).the number of shoot Differentia -tion is best of all;which was placed on rootage medium(MSi/2+0.2mg/L IBA),the rootage effect is very fine,and gains more regeneration plants.2. The genetic transformatin system has been established.The suitable antibiotics and their concentration were studied for the selecting of transformants. The suitable concentration of the filter, Kanamicin is lOmg/L; The suitable concentration of Cefazolium natriun, the most effective antibiotics,is 300~500mg/L.The genotype is a main factor in the genetic transformation via agrobacteriwn -mediated.The results of orthogonal experiments of the factors which affect mainly the transformation illuminates that the ErNiuXin genotype +bacterium 15 X +explant+dip-dye is the best one .The cocultivation time is 28 days.The experiment shows that:The ErNiuXin cotyledon regeneration frequency is very high, and that the Dongnong901 hypocotyl regeneration frequency is very high.The influence of different strain. The ability of invasion of R1000 was much better than EHA105 when comparing the number of resistant buds.But,the number of transformationpanlt ,R 1000 was lower than EHA105.3. Chinese cabbage resistant virus transgenic plant have been obtained.Transgenic T0 progeny were checked by PCR and PCR-Southern methods,The results prove the existence of target genes in transformation plants. 15 masculine plants, but RT-PCR check,only!2 plants express. The phenomenon of gene silence existed in the part of transgenic plants. The segregation proportion of target genes in transgenic Tl progeny is 3:1 .accordant with the law of Mendelian segregation in a single loci.4. Meet virus experimentThe virus inoculation experiment suggests that the disease incidence and disease index of transgenic Tl progeny were lower than those of CK.Name:Ma WeiMajonCrop Genetics and Breeding Tutor:Prof.ChuiChongShi更多还原