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中文题名:

 农杆菌介导的黄瓜遗传转化体系优化及T-DNA插入突变体的创制与鉴定    

姓名:

 冯路路    

学号:

 2017104047    

保密级别:

 公开    

论文语种:

 chi    

学科代码:

 090202    

学科名称:

 农学 - 园艺学 - 蔬菜学    

学生类型:

 硕士    

学位:

 农学硕士    

学校:

 南京农业大学    

院系:

 园艺学院    

专业:

 蔬菜学    

研究方向:

 遗传育种与生物技术    

第一导师姓名:

 陈劲枫    

第一导师单位:

 南京农业大学    

完成日期:

 2020-04-14    

答辩日期:

 2020-06-06    

外文题名:

 Optimization of Cucumber Genetic Transformation System Mediated by Agrobacterium and Creation and Identification of T-DNA Insertion Mutants    

中文关键词:

 黄瓜 ; 遗传转化 ; 体系优化 ; T-DNA ; TAIL-PCR    

外文关键词:

 cucumber ; genetic transformation ; system optimization ; T-DNA ; TAIL-PCR    

中文摘要:

黄瓜( Cucumis sativus L.)是葫芦科甜瓜属的一种重要的园艺和经济作物,具有悠久的栽培历史。在生产中由于生物胁迫和非生物胁迫的存在,对栽培黄瓜的产量、品质都造成很大程度的影响,因此为保障黄瓜产量和品质需要不断进行选育黄瓜新品种。然而传统黄瓜育种方式由于其育种年限长、抗性基因较少、效率较低等因素,很难快速得到满足人们需求的优良品种。转基因技术是现代分子育种的重要手段,其在解析基因功能、基因工程育种以及基因组编辑等方面都发挥着重要作用。利用黄瓜遗传转化进行新品种选育可以克服传统黄瓜育种的多重困扰。农杆菌介导的遗传转化法是黄瓜转基因技术中应用最为普遍和广泛的,但目前该方法的转化效率仍待进一步优化。本研究从农杆菌自身活力、共培养温度、农杆菌侵染液浓度和共培养时间等方面对农杆菌介导的黄瓜遗传转化体系进行优化,通过数据统计确定了最优转化条件。

随着黄瓜研究在分子水平上的深入发展,越来越多具有重要功能的基因被定位并克隆出来,基因功能的研究也愈发重要。突变体与其相应的突变基因之间密切相关,可以直接有效的用于基因功能研究。而黄瓜因其遗传基础狭窄、自然变异频率很低,自发突变体的获得概率极低。因此人为创制突变体进行重要功能基因研究逐渐成为趋势。突变体创制方法包括物理诱变、化学诱变以及插入突变等多种方法,插入突变中的T-DNA插入突变具有可以研究无表型的基因、易得到插入突变位点、突变位点较少等多个优点,对于突变体的创制及基因功能研究较为适合。本研究利用GFP报告基因对黄瓜遗传转化体系进行优化,并进行黄瓜T-DNA插入突变体的创制工作。研究结果如下:

1. 利用GFP基因农杆菌介导的黄瓜遗传转化体系优化 

在本实验室已经建立的农杆菌介导的黄瓜子叶节遗传转化体系的基础上,通过测定农杆菌C58的生长曲线,确定选取培养12~18 h的菌液制备侵染液。从共培养温度、共培养时间和农杆菌侵染液浓度等方面,对黄瓜遗传转化体系进行优化。结果发现,当共培养温度为23 ℃时,外植体再生率达到最高的31.70%,阳性诱导率达到最高的2.26%;将侵染液浓度和共培养时间联合处理,当侵染液OD值为0.1和共培养时间为7 d时,外植体再生率达到最高的40.13%,而不同处理的外植体阳性诱导率并没有显著差异。综上,本研究得到了选取培养12-18 h的菌液制备侵染液,共培养温度为23 ℃的优化的遗传转化体系。

2. 黄瓜T-DNA插入突变体的创制及快速鉴定

利用优化的黄瓜遗传转化体系,以pGreen0029植物表达载体为T-DNA插入载体进行黄瓜T-DNA插入突变体的创制。利用GFP荧光鉴定、PCR和TAIL-PCR方法对T0代植株进行鉴定。利用GFP荧光观察在外植体分化阶段就开始进行阳性植株筛选,从2978个转化外植体中筛选出3株表达GFP信号的阳性芽;PCR鉴定结果表明T0代转化植株的基因组中整合了载体序列;利用TAIL-PCR方法进行T-DNA侧翼序列扩增,初步确定了突变体T-DNA-PG-1的T-DNA插入位点;根据T-DNA插入位点设计特异性引物进行PCR检测,对TAIL-PCR结果得到证实。综上,本研究得到了成功转入T-DNA载体的黄瓜突变体,并通过TAIL-PCR技术确定了黄瓜突变体T-DNA-PG-1的突变位点。

外文摘要:

Cucumber (Cucumis sativus L.) is an important horticultural and economic crop in the genus Cucurbitaceae of Cucurbitaceae, with a long history of cultivation. Due to the existence of biotic and abiotic stress in production, it has a great impact on the yield and quality of cultivated cucumbers. Therefore, in order to ensure the yield and quality of cucumbers, new cucumber varieties need to be continuously selected. However, due to factors such as long breeding years, few resistance genes, and low efficiency, the traditional cucumber breeding methods make it difficult to quickly obtain excellent varieties that meet people's needs. Transgenic technology is an important means of modern molecular breeding, and it plays an important role in analyzing gene function, genetic engineering breeding and genome editing.Transgenic technology is an important means of modern molecular breeding. The use of genetic transformation of cucumber to select new varieties can overcome the multiple problems of traditional cucumber breeding. The Agrobacterium-mediated genetic transformation method is the most widely used and widely used in cucumber transgenic technology, but the transformation efficiency of this method still needs to be further optimized. This study optimized the Agrobacterium-mediated transformation system of cucumber genetic transformation from aspects of Agrobacterium's own vitality, co-cultivation temperature, Agrobacterium infectious solution concentration, and co-cultivation time. The optimal relevant transformation conditions were determined through data statistics.

With the further development of cucumber research at the molecular level, more and more genes with important functions have been located and cloned, and the study of gene functions has become increasingly important. Mutants are closely related to their corresponding mutant genes and can be directly and effectively used for gene function research. However, because of its narrow genetic basis and low natural mutation frequency, the probability of obtaining spontaneous mutants is extremely low. Therefore, artificially creating mutants for research on important functional genes has gradually become a trend. Mutation creation methods include physical mutagenesis, chemical mutagenesis, and insertion mutations. T-DNA insertion mutations in insertion mutations have many advantages such as the ability to study non-phenotypic genes, easy access to insertion mutation sites, and fewer mutation sites. They are suitable for the creation of mutants and gene functions. In this study, the GFP reporter gene was used to optimize the cucumber genetic transformation system, and the creation of cucumber T-DNA insertion mutants was performed.The findings are as follows:

1. Optimization of cucumber genetic transformation system using GFP geneOptimization of cucumber genetic transformation system mediated by agrobacterium

Based on the Agrobacterium-mediated genetic transformation system of cucumber cotyledonary nodes established in this laboratory, by measuring the growth curve of Agrobacterium C58, it was determined that the bacterial solution cultured for 12-18 hours was selected to prepare the infectious solution. The genetic transformation system of cucumber was optimized from the aspects of co-cultivation temperature, co-cultivation time and the concentration of Agrobacterium infection solution. The results showed that when the co-cultivation temperature was 23 ° C, the explant regeneration rate reached the highest 31.70%, and the positive induction rate reached the highest 2.26%; the concentration of the infecting solution and the co-cultivation time were combined to be treated. When it was 0.1 and the co-cultivation time was 7 days, the explant regeneration rate reached the highest 40.13%, while the positive induction rate of explants under different treatments was not significantly different. In summary, in this study, an optimized genetic transformation system was obtained in which the inoculum was selected from the bacterial solution cultured for 12-18 hours, and the co-cultivation temperature was 23 ° C.

2. Creation and rapid identification of cucumber T-DNA insertion mutants

Using the optimized cucumber genetic transformation system, pGreen0029 plant expression vector was used as a T-DNA insertion vector to create a cucumber T-DNA insertion mutant. To plants were identified by GFP fluorescence identification, PCR and TAIL-PCR. GFP fluorescence observation was used to screen for positive plants at the stage of explant differentiation, three positive shoots expressing GFP signals were screened out of 2978 transformed explants. PCR identification results showed that the T0 transformed plants had integrated vector sequences. T-DNA flanking sequence amplification using the TAIL-PCR method, the T-DNA insertion site of the mutant T-DNA-PG-1 was initially determined.  Designing specific primers based on the T-DNA insertion site further confirmed the accuracy of the T-DNA insertion site. The comprehensive results showed that the pGreen vector was transferred into the cucumber genome, and the insertion position of T-DNA in the cucumber genome was determined by the T-DNA flanking sequence amplification.

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中图分类号:

 S63    

开放日期:

 2020-06-16    

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