發(fā)布時(shí)間：2018-04-22 00:08

  本文選題：基因編輯 + 水稻��； 參考：《國(guó)家海洋局第三海洋研究所》2017年碩士論文

【摘要】：第一部分水稻基因編輯技術(shù)體系建立及深海細(xì)菌醛脫氫酶的水稻轉(zhuǎn)化研究水稻是一種重要的糧食作物,獲得優(yōu)良的水稻品種對(duì)農(nóng)業(yè)發(fā)展具有重要意義。新興的CRISPR/Cas9基因編輯技術(shù)在近幾年內(nèi)飛速發(fā)展,其構(gòu)建方法簡(jiǎn)單、打靶效率高以及基因編輯的靶向性使得它在育種工作中具有極大的應(yīng)用潛力。在植物育種尋求新突破的探索中,深海微生物中具有特殊功能的極端酶基因可能成為功能基因的巨大寶庫(kù),將這些酶基因?qū)胫参飪?nèi),可能可以對(duì)植物的生長(zhǎng)以及抗逆性等方面產(chǎn)生積極的影響或可將植物變?yōu)樯a(chǎn)極端酶的“工廠”。本研究利用水稻建立基因編輯體系,首先使用CRISPR/Cas9技術(shù)對(duì)控制水稻分蘗性狀中的關(guān)鍵調(diào)控基因D27進(jìn)行定點(diǎn)敲除,成功獲得了突變體植株,在T0代植株基因的靶位點(diǎn)附近檢測(cè)出了堿基的插入、替換和缺失,并使得突變體植株葉部D27的表達(dá)量較野生型明顯下降,同時(shí)T1代突變體植株出現(xiàn)了明顯的矮化、多分蘗以及葉片變窄變短的表型變化。接著,選取對(duì)植物抗逆性緊密相關(guān)且具有醛類物質(zhì)解毒害作用的醛脫氫酶作為突破口,將深海來(lái)源的鹽單胞菌Halomonas axialensis中的醛脫氫酶基因通過(guò)農(nóng)桿菌介導(dǎo)的轉(zhuǎn)化技術(shù)導(dǎo)入水稻基因組,在獲得的轉(zhuǎn)基因愈傷組織中檢出轉(zhuǎn)入的外源醛脫氫酶基因已成功表達(dá)。本實(shí)驗(yàn)對(duì)深海微生物資源在植物中的開發(fā)利用做出了初步的探索,并利用CRISPR/Cas9技術(shù)初步建立了后期轉(zhuǎn)基因功能驗(yàn)證的評(píng)價(jià)體系。第二部分深海海水中間苯二甲酸二甲酯降解菌的多樣性分析由于塑料制品的廣泛使用和大量生產(chǎn),鄰苯二甲酸酯類(PAEs)化合物的污染已遍布全球。研究表明,PAEs對(duì)生物體具有致癌致畸、造成組織損傷等危害,同時(shí)也是一類環(huán)境激素,對(duì)動(dòng)物的生殖及胎兒發(fā)育具有諸多危害。因此PAEs的污染狀況亟待治理。目前PAEs類化合物降解方式主要分為非生物降解的水解和光解以及生物降解兩種,由于自然條件下的水解和光解方式的降解速率極慢,因此生物降解是PAEs類降解的主要方式。本研究選取PAEs類化合物中的間苯二甲酸二甲酯(DMI)做為降解底物,在我國(guó)南海2000 m深海處分別采集原位富集和實(shí)驗(yàn)室富集兩種樣品,通過(guò)未培養(yǎng)和實(shí)驗(yàn)室培養(yǎng)兩種方式對(duì)深海海水中DMI降解菌的多樣性進(jìn)行分析。聚類分析結(jié)果顯示,原物富集與實(shí)驗(yàn)室富集樣品中的優(yōu)勢(shì)菌群有較大不同,推測(cè)這種現(xiàn)象是由于兩種富集方式中海水含氧量的不同造成的。同時(shí)由于培養(yǎng)方式和培養(yǎng)條件的局限性,未培養(yǎng)和實(shí)驗(yàn)室培養(yǎng)方式獲得的優(yōu)勢(shì)菌群也存在較大差異。本實(shí)驗(yàn)中通過(guò)不同的富集方式和培養(yǎng)方式共分析得到優(yōu)勢(shì)菌株Sulfurovum、Corynebacterium、Thalassospira等8個(gè)屬,其中大部分菌株均有報(bào)道稱可降解多環(huán)芳烴或石油等物質(zhì)。上述菌株的獲得對(duì)DMI污染的治理提供了一定的參考價(jià)值。
[Abstract]:The first part is the establishment of rice gene editing technique system and the transformation of deep-sea bacterial aldehyde dehydrogenase. Rice is an important food crop. It is of great significance to obtain good rice varieties for agricultural development. With the rapid development of CRISPR/Cas9 gene editing technology in recent years, its simple construction method, high efficiency of targeting and targeting of gene editing make it have great application potential in breeding work. In the search for new breakthroughs in plant breeding, extreme enzyme genes with special functions in deep-sea microbes may become a huge repository of functional genes, which can be introduced into plants. It may have a positive effect on plant growth and resistance, or turn plants into "factories" that produce extreme enzymes. In this study, rice gene editing system was established. Firstly, CRISPR/Cas9 technique was used to knockout the key regulatory gene D27 in rice tillering traits, and the mutant plant was successfully obtained. The insertion, substitution and deletion of the bases were detected near the target site of the gene of generation T0. The expression of D27 in the leaves of the mutant was significantly lower than that in the wild type, and the plant of the T1 generation was obviously dwarfed. Phenotypic changes of multiple tillers and narrow and shorter leaves. Then, aldehydes dehydrogenase, which is closely related to plant stress resistance and has the ability to detoxify aldehydes, is chosen as the breakthrough point. Aldehyde dehydrogenase gene from deep-sea Halomonas axialensis was introduced into rice genome by Agrobacterium tumefaciens mediated transformation, and the transferred aldehydes dehydrogenase gene was successfully expressed in transgenic callus. This experiment made a preliminary exploration on the exploitation and utilization of deep-sea microbial resources in plants, and established a preliminary evaluation system for the validation of transgenic functions in the later stage by using CRISPR/Cas9 technology. The second part the diversity analysis of dimethyl phthalate degradation bacteria in deep-sea seawater. Due to the widespread use and mass production of plastic products the pollution of phthalate ester PAEs-based compounds has spread all over the world. Studies have shown that PAEs have carcinogenic teratogenicity, tissue damage and other hazards, but also a class of environmental hormones, which has a lot of harm to the reproduction and fetal development of animals. Therefore, the pollution of PAEs is in urgent need of treatment. At present, the degradation methods of PAEs compounds are mainly divided into non-biodegradable hydrolysis, photolysis and biodegradation. Because the degradation rate of natural hydrolysis and photolysis is very slow, biodegradation is the main way of PAEs degradation. In this study, dimethyl isophthalate (Dimethyl isophthalate) from PAEs compounds was selected as the degradation substrate, and two samples were collected in situ enrichment and laboratory enrichment at the depth of 2000 m in the South China Sea. The diversity of DMI degrading bacteria in deep sea water was analyzed by uncultured and laboratory culture. The results of cluster analysis show that the enrichment of raw material is different from the dominant flora in the sample enriched in laboratory. It is speculated that this phenomenon is caused by the difference of oxygen content in seawater between the two enrichment modes. At the same time, because of the limitation of culture methods and culture conditions, the dominant flora obtained by uncultured and laboratory culture was also different. In this experiment, 8 genera of dominant strain Sulfurovum Corynebacterium Thalassospira were obtained by different enrichment and culture methods. Most of the strains were reported to be degradable polycyclic aromatic hydrocarbons (PAHs) or petroleum and other substances. The above-mentioned strains provide some reference value for the control of DMI pollution.
【學(xué)位授予單位】：國(guó)家海洋局第三海洋研究所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S511;Q943.2
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本文編號(hào)：1784708