以高品质为导向的半夏基因工程研究

发布时间：2018-05-18 09:25

本文选题：半夏 + 根癌农杆菌　；参考：《华中农业大学》2017年硕士论文

【摘要】：半夏(Pinellia ternata(Thunb.)Breit)为天南星科半夏属植物,是传统名贵中药材。半夏块茎是中医化痰燥湿、止痛消肿、止吐降逆的良药。半夏在全国各地均有分布,虽分布广泛,但由于过度开采和缺乏科学规范的种植技术,半夏优良种质资源面临稀缺。同时,针对半夏的分子遗传转化方面的研究还比较少,分子育种较为欠缺。为了获得兼具药用成分高、抗逆性和抗病性强的半夏新品种,本研究以荆半夏为材料,利用半夏一体成苗技术获得大量无菌苗;然后通过优化的农杆菌介导转化体系,将来源于拟南芥(Arabidopsis thaliana)的抗热蛋白ERECTA(ER)、来源于金黄色葡萄球菌(Staphylococcus aureus)有助于提高生物碱表达量的高活性3-羟基-3-甲基戊二酰还原辅酶A(3-hydroxy-3-methylglutaryl-Co Areduct-as e,HMGR)、来源于甜菜夜蛾(Spodoptera exigua)有助于提高植物抗逆能力的凋亡抑制因子(Inhibitor of apoptosis proteins,IAPs)、抗除草剂双丙氨酰磷抗性蛋白(Bioiaphos resistance,BAR)等编码基因转化到半夏植株中,获得基因组整合的半夏植株。同时构建HMGR和ER基因双价高表达载体,让这两个基因在同一株半夏中有较高的表达。通过农杆菌转化后上述转基因体系共得到301株抗性苗,提取基因组DNA,PCR验证后得到72株阳性苗随后利用实时荧光定量PCR(Real Time PCR)技术检测基因在半夏中的表达量,发现转基因半夏中几种基因的表达量均有了较大的提高,证明上述基因均在半夏中呈高水平表达。
[Abstract]:Pinellia ternata (Thunb. Breititi) is a traditional and precious traditional Chinese medicinal material, belonging to Pinellia ternata, a genus of Pinellia ternata. The tuber of Pinellia ternata is a good medicine for relieving phlegm and dampness, relieving pain and swelling, and reducing vomiting and adverse effects. Pinellia ternata is widely distributed all over the country, but due to over-exploitation and lack of scientific and standardized planting technology, the excellent germplasm resources of Pinellia ternata are scarce. At the same time, the research on molecular genetic transformation of Pinellia ternata is less, and molecular breeding is less. In order to obtain a new variety of Pinellia ternata with high medicinal composition, high resistance to stress and disease resistance, a large number of sterile seedlings were obtained by using Pinellia ternata as a material, and then the transformation system was optimized by Agrobacterium tumefaciens. The high activity 3-hydroxy-3-methyl-glutaryl reductive coenzyme A(3-hydroxy-3-methylglutaryl-Co Areduct-as ehmgra from Arabidopsis thaliana and Staphylococcus aureus from Staphylococcus aureus, which is derived from Spodoptera exigua, is helpful. The inhibitor of apoptosis proteinsi, a inhibitor of plant stress resistance, and Bioiaphos resistance1, a herbicide resistant protein, were transformed into Pinellia ternata plants. Genomic integration of Pinellia ternata was obtained. The bivalent expression vector of HMGR and ER gene was constructed at the same time, and the two genes were highly expressed in the same strain of Pinellia ternata. A total of 301 resistant seedlings were obtained by Agrobacterium tumefaciens transformation, and 72 positive seedlings were obtained after genomic DNA-PCR verification. The expression of the gene in Pinellia ternata was detected by real-time fluorescence quantitative PCR(Real Time. It was found that the expression of several genes in the transgenic Pinellia ternata was greatly improved, which proved that the above genes were highly expressed in Pinellia ternata.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S567.239

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