CRISPR-Cas系统对老汉瓜ACC合成酶基因定点敲除及功能验证

发布时间：2018-05-05 22:18

本文选题：CRISPR-Cas9 + 基因敲除　；参考：《新疆大学》2017年硕士论文

【摘要】：甜瓜(Cucumis melo L.)属葫芦科黄瓜属,是一种深得人们喜爱的水果,而“老汉瓜”维吾尔语称之为“比歇克吉”,学名为歇克甜瓜,是新疆特产,具有柔软可口、水分多、糖度高等特点,受到广大消费者欢迎。由于老汉瓜成熟以后,其软化的速度很快,对储藏和运输造成了很大的困难,使经济受到了很大的损失。因此,提高果实的耐储藏性成为生产上必须要解决的难题,也是甜瓜育种的重要目标。随着科学技术的发展,人们发现乙烯可以控制果实的成熟衰老,而促进乙烯合成的主要限速酶是ACC合成酶,这个发现成为了改善甜瓜储藏性的理论依据。CRISPR/Cas系统是新一代基因编辑系统,可以用来靶向敲除或敲入目的基因。因此本实验以老汉瓜为实验材料,以甜瓜ACC合成酶基因为基础设计引物,根据CRISPR-Cas9系统构建敲除甜瓜ACC合成酶基因的表达载体,然后通过农杆菌对“老汉瓜”进行遗传转化,从而得到阳性转基因甜瓜T0代,以此为材料进行生理生化检测。主要结果如下:1.以ACS基因序列为基础,设计靶位点,将合成的引物构建到植物基因敲除载体p P1C.4上,获得了敲除甜瓜ACC合成酶基因的表达载体p P1C.4-ACS。2.对甜瓜品种“老汉瓜”的组织培养体系进行了探索和优化,确定了适合的培养体系。通过农杆菌GV3101进行甜瓜的遗传转化,获得了3株T0代的转基因甜瓜苗。3.观察转化前后甜瓜苗的形态变化,发现敲出ACC合成酶基因后,抑制了根部的伸长,并且延缓了植株的生长和黄化。对转基因甜瓜的SOD、POD和CAT的酶活性进行检测,发现这三种酶的活性均高于野生型甜瓜,初步验证了ACC合成酶对植物衰老和对逆境的耐受能力起着重要作用。同时为进一步研究敲除ACC合成酶对甜瓜果实成熟的影响奠定基础。
[Abstract]:Melon (Cucumis melo L.) belongs to Cucumis Cucumis, a kind of fruit which is deeply loved by people. "Old Gua" Uygur language is called "bickgi". It is called "chic" melon. It is a special product of Xinjiang. It has the characteristics of soft and tasty, more water and high sugar. It is welcomed by many consumers. Because old Chinese melon is mature, its softening speed is fast. As a result of the development of science and technology, it is found that ethylene can control the maturity of fruit and promote the synthesis of ethylene with the development of science and technology. The main speed limiting enzyme is the ACC synthetase. This discovery has become a theoretical basis for improving the storage of melon. The.CRISPR/Cas system is a new generation of gene editing system, which can be used to target knocking or knocking on the target gene. Therefore, the old Chinese melon was used as the experimental material to design primers based on the base of melon ACC synthetase, according to the CRISPR-Cas9 system. The expression vector that knocks the ACC synthase gene of melons is constructed, and then genetically transformed by Agrobacterium tumefaciens to the "old Chinese melon", and then the T0 generation of the positive transgenic melon is obtained. The main results are as follows: 1. based on the ACS gene sequence, the target loci are designed and the primers are constructed to plant gene knockout. On the carrier P P1C.4, the expression vector of the ACC synthase gene of melon was knocked out, P P1C.4-ACS.2. was used to explore and optimize the tissue culture system of the melon variety "old Chinese melon", and the suitable culture system was determined. The genetic transformation of muskmelon by Agrobacterium GV3101 was carried out, and 3 transgenic melon seedlings of T0 generation were obtained before the transformation of.3.. The morphological changes of the later sweet melon seedlings showed that after the ACC synthase gene was knocked out, the elongation of the roots was inhibited and the growth and yellowing of the plants were delayed. The enzyme activities of SOD, POD and CAT of the transgenic melon were detected, and the activity of these three enzymes was higher than that of the wild type melon, and the initial step proved that the ACC synthase was resistant to plant senescence and resistance to adversity. It is important to further study the effects of knockdown of ACC synthase on melon ripening.

【学位授予单位】：新疆大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S652;Q943.2

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