基因工程改造小球藻提高其虾青素表达量

发布时间：2018-05-12 03:16

本文选题：酮化酶 + 小球藻　；参考：《合肥工业大学》2017年硕士论文

【摘要】：虾青素是一种次生类胡萝卜素,具有超强的抗氧化性,在生物体内能淬灭单线态氧、清除自由基,可以防止机体衰老,增强免疫力。同时还具有抗癌抑癌、预防心血管疾病等生物学功能,广泛应用于食品、医药和保健品等行业。小球藻含有虾青素,具有生长速度快、生物密度高、易培养等优点,是很好的生物反应器。但是小球藻中虾青素含量低,应用基因工程的方法提高虾青素含量成为近年来研究的热点。农杆菌具有操作简单、转化效率高、经济实用等优点,近年来广泛应用于藻类的遗传转化。本研究建立农杆菌介导的小球藻遗传转化的方法,构建β-胡萝卜素酮化酶基因表达载体,并通过农杆菌介导的方法转入小球藻中,提高虾青素含量。本论文具体研究内容和成果如下:1、筛选体系的确定:研究羧苄青霉素对农杆菌和小球藻的影响,羧苄青霉素浓度为200mg/L时抑制农杆菌的生长,羧苄青霉素对小球藻生长无影响。研究小球藻在BBM液体培养基和固体培养基中对G418的敏感性,确定在BBM液体培养中G418的筛选浓度为1mg/L;在BBM固体培养基中G418的筛选浓度为2mg/L。最终确定BBM液体培养基中G418浓度为1mg/L和羧苄青霉素浓度为500mg/L的小球藻筛选体系;在固体培养基中G418浓度为2mg/L和羧苄青霉素浓度为500mg/L的小球藻筛选体系。2、以GFP为报告基因,以G418抗性基因(NPT II)作为筛选标记基因,CaMV35S为启动子,PBI121为载体,利用农杆菌介导转化的方法,建立小球藻遗传转化体系。通过PCR验证,GFP基因在小球藻内完成基因重组;通过RT-PCR验证,GFP基因成功转录;在激光共聚焦下转化子显示绿色荧光,GFP基因成功翻译。转化200天后,阳性鉴定依然为阳性,GFP稳定遗传表达。3、构建PBI121::35S::PDSSP-BKT载体。PDSSP为调控八氢番茄红素的信号肽基因,BKT为β-胡萝卜素酮化酶基因,先通过overlap PCR技术克隆获得PDSSP-BKT融合基因。4、利用农杆菌介导转化的方法,将PDSSP-BKT融合基因转入小球藻,并筛选阳性转化子,提取虾青素,通过HPLC测定,在相同培养条件下,转化子中虾青素含量比野生型高63.5%。
[Abstract]:Astaxanthin (astaxanthin) is a kind of secondary carotenoid. It can quench singlet oxygen and scavenge free radical in organism. It can prevent aging and enhance immunity. It also has the biological functions of anticancer and cancer suppression, cardiovascular disease prevention and so on. It is widely used in food, medicine and health products. Chlorella microphylla contains astaxanthin, which has the advantages of fast growth rate, high biological density and easy to culture, so it is a good bioreactor. However, the content of astaxanthin in Chlorella vulgaris is low, and the application of genetic engineering to improve the content of astaxanthin has become a hot topic in recent years. Agrobacterium tumefaciens has been widely used in genetic transformation of algae in recent years because of its advantages of simple operation, high efficiency, economy and practicality. In this study, a method of Agrobacterium tumefaciens mediated genetic transformation of Chlorella vulgaris was established, and 尾 -carotene ketonase gene expression vector was constructed and transferred into Chlorella vulgaris by Agrobacterium tumefaciens to increase astaxanthin content. The specific contents and results of this paper are as follows: 1. The effects of Carbenicillin on Agrobacterium tumefaciens and Chlorella vulgaris were studied. When the concentration of Carbenicillin was 200mg/L, the growth of Agrobacterium tumefaciens was inhibited, but Carbenicillin had no effect on the growth of Chlorella microphylla. The sensitivity of Chlorella vulgaris to G418 in BBM liquid medium and solid medium was studied. The screening concentration of G418 in BBM liquid culture was 1 mg / L and that in BBM solid medium was 2 mg / L. The screening system of Chlorella vulgaris with G418 concentration of 1mg/L and Carbenicillin concentration of 500mg/L in BBM liquid medium and Chlorella vulgaris screening system with G418 concentration of 2mg/L and Carbenicillin concentration of 500mg/L in solid medium were determined. GFP was used as reporter gene. The genetic transformation system of Chlorella microphylla was established by Agrobacterium tumefaciens mediated transformation with G418 resistant gene NPT II as a screening marker gene CaMV35S as promoter PBI121. The GFP gene was recombined in Chlorella vulgaris by PCR, the GFP gene was successfully transcribed by RT-PCR, and the green fluorescent GFP gene was successfully translated by confocal laser. After 200 days of transformation, the positive identification was still positive for the stable genetic expression of GFP. PBI121::35S::PDSSP-BKT vector. PDSSP was constructed as the signal peptide gene to regulate the octyllycopene. BKT was 尾 -carotene ketone enzyme gene. The PDSSP-BKT fusion gene. 4 was cloned by overlap PCR technique. The PDSSP-BKT fusion gene was transferred into Chlorella vulgaris by Agrobacterium tumefaciens, and the positive transformants were screened. Astaxanthin was extracted by HPLC assay under the same culture conditions. The content of astaxanthin in transformants was 63.5% higher than that in wild type.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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