摘要
目前,因为转化效率的问题,大多数植物遗传转化操作需要使用选择标记基因,诸如抗生素或除草剂抗性基因等来筛选转化子,虽然仍无直接研究结果表明选择标记基因影响人类健康或环境安全,但近年来人们对转基因食品安全性的担心却与日俱增,为了消除转基因食品的安全性顾虑,无选择标记转基因植物应运而生。本文初步对Cre/lox重组系统进行了研究,并用已构建好的能有效删除标记基因的Cre/loxp组成型载体利用农杆菌介导转化烟草,获得了共转化烟草植株;植物异黄酮是一类具有增强植物抗病、诱导根瘤形成以及预防激素相关肿瘤发生、缓解女性更年期综合症的活性次生代谢产物,其合成关键酶是异黄酮合酶(IFS);本研究为了改良加工番茄的品质特性,使人们在食用加工番茄产品的同时获得更好的保健效果,对加工番茄遗传转化的再生体系进行了优化,构建了含有IFS基因的植物表达载体pBI121-ifs,并用根癌农杆菌介导转化加工番茄子叶且获得了转基因植株;果树由于自身的特点,使得遗传转化有很大的难度和特殊性。所以本文以草莓和苹果为试材,建立了适用于草莓和苹果转基因的高效遗传转化体系。
本研究的主要结论如下:
1.本试验对获得的22株共转化烟草叶片提取DNA进行PCR检测,结果证明有10株成功删除了gus基因,删除率为45.5%;
2.农杆菌介导转化加工番茄时筛选出的最适宜培养基:预培养培养基是MS+B5+IAA1.0mg/L+6-BA2.0mg/L,筛选转化苗培养基为MS+B5+IAA1.0mg/L+6-BA2.0 mg/L+Cef500mg/L+Kan50mg/L ,转化苗生根培养基为l/2MS+B5+IBA2.0mg/L+ Cef300mg/L+Kan50mg/L,对获得的11株转化加工番茄提取DNA和RNA进行PCR和RT-PCR检测,结果证明有4株已经成功转入了ifs基因,转化效率为36.4%;
3.草莓离体再生体系建立中筛选的最佳培养基:茎尖快繁培养基l/2MS+B5+NAA0.5mg/L+TDZ0.5mg/L,不定芽诱导培养基MS+B5+NAA0.3mg/L+TDZ3.0 mg/L;
苹果离体再生体系建立中筛选的最佳培养基:茎尖快繁培养基为l/2MS+B5+NAA0.2mg/L+6-BA 1.0 mg/L ,不定芽诱导培养基为MS+B5+ NAA0.1mg/L+ZT1.0 mg/L。
上述研究可为今后进一步获得安全性的转基因果实奠定一定的技术基础。
At present, most of the transgenic plants use the selectable marker genes such as antibiotic and herbicide resistanse to select transplants.Recent years,more and more people worry about the safety of transgenic product.Although there is no report shows that selectable marker genes can affect the human health and the environment security.Marker-free transgenic system is a rapidly developed method which can eliminate the safty concern of transgenic food.This paper studied on Cre/loxp recoganization system, and agrobacterium-mediated transformation of tobacco with Cre/loxp system which constructed can delete the marker genes, and obtained the co-transformation tobacco plant.Isoflavones are a group of secondary metabolites common to leguminous plants that play roles in plant defense and nodulation and have many benefits to human health including relief of menopausal symptoms,reduction of osteoporosis and lowering risk of certain hormone-related cancers. Isoflavone synthase(IFS),which is the key enzyme in isoflavone biosynthesis. This study optimizated the transgenetic regeneration system of tomato, Constructed the pBi121-ifs verctor, and Agrobacterium-mediated transformation of processing tomato as well as obtained the transgenetic plant. The fruit tree have certain difficulty and particularity in genetic transformation. To obtain highly effective genetic transformation method about strawberry and apple, the optimization of regeneration System of strawberry leaf and apple leaf were established.
The study includes three major conclusions as followed:
1. In this experiment ,DNA was extracted from co-transformation tobacco leaves of 22 plants and was detected by PCR.The results showed that gus gene had been deleted successfully from 10 plants and delete rate was 45.5%.
2. Agrobacterium-mediated transformation of processing tomato: The pre-culture medium was MS+B5 supplied with IAA1.0 mg/L +6-BA2.0mg/L,The high efficient media for selection was MS+B5 supplied with IAA1.0 mg/L +6-BA 2.0 mg/L +Cef500 mg/L +Kan50mg/L,The high efficient media for root induction was MS+B5 supplied with IBA2.0 mg/L + Cef300 mg/L +Kan50mg/L, DNA and RNA was extracted from the 11 Genetic Transformation of Processing Tomatoes and detected by PCR and RT-PCR.the results showed that ifs gene had been successfully transfered into 4 plants and the transformation efficiency was 36.4%.
3. Establishment of regeneration system of strawberry: The best effects of stalk culture in medium was l/2(MS+B5) supplied with NAA0.5mg/L +TDZ0.5 mg/L,The best effects of buds induction culture in medium was MS+B5 supplied with NAA0.3 mg/L+TDZ3.0 mg/L,Establishment of regeneration system of strawberr apple:The best effects of stalk culture in medium was l/2(MS+B5) supplied withNAA0.2mg/L +6-BA 1.0 mg/L,The best effects of buds induction culture in medium was MS+B5 supplied with NAA0.1mg/L+ZT1.0 mg/L.
The research above provided the technology foundation for obtaining the transgene-safty fruits in the future.
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