本文選題：CRISPR-Cas9 + 基因敲除�。� 參考：《新疆大學(xué)》2017年碩士論文

【摘要】：甜瓜(Cucumis melo L.)屬葫蘆科黃瓜屬,是一種深得人們喜愛的水果,而“老漢瓜”維吾爾語(yǔ)稱之為“比歇克吉”,學(xué)名為歇克甜瓜,是新疆特產(chǎn),具有柔軟可口、水分多、糖度高等特點(diǎn),受到廣大消費(fèi)者歡迎。由于老漢瓜成熟以后,其軟化的速度很快,對(duì)儲(chǔ)藏和運(yùn)輸造成了很大的困難,使經(jīng)濟(jì)受到了很大的損失。因此,提高果實(shí)的耐儲(chǔ)藏性成為生產(chǎn)上必須要解決的難題,也是甜瓜育種的重要目標(biāo)。隨著科學(xué)技術(shù)的發(fā)展,人們發(fā)現(xiàn)乙烯可以控制果實(shí)的成熟衰老,而促進(jìn)乙烯合成的主要限速酶是ACC合成酶,這個(gè)發(fā)現(xiàn)成為了改善甜瓜儲(chǔ)藏性的理論依據(jù)。CRISPR/Cas系統(tǒng)是新一代基因編輯系統(tǒng),可以用來靶向敲除或敲入目的基因。因此本實(shí)驗(yàn)以老漢瓜為實(shí)驗(yàn)材料,以甜瓜ACC合成酶基因?yàn)榛A(chǔ)設(shè)計(jì)引物,根據(jù)CRISPR-Cas9系統(tǒng)構(gòu)建敲除甜瓜ACC合成酶基因的表達(dá)載體,然后通過農(nóng)桿菌對(duì)“老漢瓜”進(jìn)行遺傳轉(zhuǎn)化,從而得到陽(yáng)性轉(zhuǎn)基因甜瓜T0代,以此為材料進(jìn)行生理生化檢測(cè)。主要結(jié)果如下:1.以ACS基因序列為基礎(chǔ),設(shè)計(jì)靶位點(diǎn),將合成的引物構(gòu)建到植物基因敲除載體p P1C.4上,獲得了敲除甜瓜ACC合成酶基因的表達(dá)載體p P1C.4-ACS。2.對(duì)甜瓜品種“老漢瓜”的組織培養(yǎng)體系進(jìn)行了探索和優(yōu)化,確定了適合的培養(yǎng)體系。通過農(nóng)桿菌GV3101進(jìn)行甜瓜的遺傳轉(zhuǎn)化,獲得了3株T0代的轉(zhuǎn)基因甜瓜苗。3.觀察轉(zhuǎn)化前后甜瓜苗的形態(tài)變化,發(fā)現(xiàn)敲出ACC合成酶基因后,抑制了根部的伸長(zhǎng),并且延緩了植株的生長(zhǎng)和黃化。對(duì)轉(zhuǎn)基因甜瓜的SOD、POD和CAT的酶活性進(jìn)行檢測(cè),發(fā)現(xiàn)這三種酶的活性均高于野生型甜瓜,初步驗(yàn)證了ACC合成酶對(duì)植物衰老和對(duì)逆境的耐受能力起著重要作用。同時(shí)為進(jìn)一步研究敲除ACC合成酶對(duì)甜瓜果實(shí)成熟的影響奠定基礎(chǔ)。
[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.

【學(xué)位授予單位】：新疆大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S652;Q943.2

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本文編號(hào)：1849524