本文選題：植物抗逆性 + 基因工程技術(shù)　； 參考：《西南科技大學(xué)》2016年碩士論文

【摘要】：耐輻射奇球菌(Deinococcus radiodurans)是迄今為止發(fā)現(xiàn)的輻射耐受性最強(qiáng)的生物之一,CFPS是位于耐輻射奇球菌R1屬的一段基因。通過NCBI數(shù)據(jù)比對發(fā)現(xiàn)CFPS與LEA76基因家族同源,屬于LEA第三族的基因。第三族LEA蛋白基因包含多個(gè)拷貝的11個(gè)氨基酸組成的基元序列(TAQAAKEKAGE)。本研究將耐輻射奇球菌CFPS全長基因分為5個(gè)結(jié)構(gòu)域,其中含有11個(gè)氨基酸重復(fù)區(qū)段的第104位至160位氨基酸結(jié)構(gòu)域,命名為CFPS-M;含有雙倍CFPS-M基因的串聯(lián)序列,命名為CFPS-2M。我們構(gòu)建了植物表達(dá)載體PCAMBIA2300-M和PCAMBIA2300-2M,分別將兩個(gè)基因轉(zhuǎn)入擬南芥中,獲得T3代轉(zhuǎn)基因陽性苗,并對其進(jìn)行了多種非生物脅迫下的抗逆性研究分析。主要結(jié)果如下:(1)成功構(gòu)建植物表達(dá)載體PCAMBIA2300-M和PCAMBIA2300-2M,并用凍融法將它們分別導(dǎo)入農(nóng)桿菌GV3103中,經(jīng)Kan抗性篩選和菌落PCR鑒定成功導(dǎo)入農(nóng)桿菌中。(2)選擇擬南芥作為受體材料,用農(nóng)桿菌介導(dǎo)法將CFPS-M和CFPS-2M基因轉(zhuǎn)入擬南芥中,經(jīng)Kan抗性篩選和對基因組DNA進(jìn)行PCR鑒定,以及實(shí)時(shí)熒光定量PCR鑒定,篩選到18株轉(zhuǎn)CFPS-M基因陽性苗和21株轉(zhuǎn)CFPS-2M基因陽性苗,并不斷繁育篩選到T3代純合陽性苗。(3)在多種非生物脅迫下,轉(zhuǎn)基因擬南芥種子的相對發(fā)芽率和相對根長都大于非轉(zhuǎn)基因植株(P0.05)。并且包含雙拷貝的CFPS-M基因的CFPS-2M轉(zhuǎn)基因擬南芥抗逆能力更強(qiáng),在干旱、鹽漬、高溫、低溫等非生物脅迫下均較為突出。(4)在多種非生物脅迫下,轉(zhuǎn)基因擬南芥成苗的生理生化指標(biāo)分析表明轉(zhuǎn)基因植株的丙二醛含量比野生型對照顯著降低(P0.05),可溶性糖、可溶性蛋白及脯氨酸等滲透調(diào)節(jié)物質(zhì)的含量顯著高于非轉(zhuǎn)基因植株(P0.05),且抗氧化酶系統(tǒng)(SOD、POD、CAT)的活性也顯著高于野生型對照(P0.05)。說明CFPS-M和CFPS-2M基因通過降低植物體內(nèi)積累的有害物質(zhì)MDA的含量及提高植物體內(nèi)滲透調(diào)節(jié)保護(hù)物質(zhì)含量、抗氧化酶系統(tǒng)的活力來提高植物抵御非生物脅迫的能力。(5)轉(zhuǎn)CFPS-2M基因擬南芥植株的抗逆性比轉(zhuǎn)CFPS-M基因植株的抗逆性更強(qiáng),說明增加了外源抗逆基因的拷貝數(shù)增強(qiáng)了該抗逆基因表達(dá)的效果,使植株有更強(qiáng)的抗逆性。
[Abstract]:Deinococcus radiodurans is one of the most radiation tolerant organisms found so far. CFPS is a gene located in the genus R1 of A. radiodurans. By comparing NCBI data, we found that CFPS is homologous to LEA76 gene family and belongs to the third family of LEA gene. The third group of LEA protein genes contain multiple copies of 11 amino acid motifs (TAQAAKEKAGEG). In this study, the full-length CFPS gene was divided into five domains, including the 104th to 160th amino acid domain with 11 amino acid repeats, and the tandem sequence containing double CFPS-M gene, named CFPS-2M. We constructed plant expression vectors PCAMBIA2300-M and PCAMBIA 2300-2Mrespectively and transferred two genes into Arabidopsis thaliana to obtain T3 generation transgenic positive seedlings. The main results were as follows: (1) the plant expression vectors PCAMBIA2300-M and PCAMBIA 2300-2M were successfully constructed, and they were introduced into Agrobacterium GV3103 by freeze-thaw method. Arabidopsis thaliana was selected as the recipient material by Kan resistance screening and colony PCR identification. CFPS-M and CFPS-2M genes were transferred into Arabidopsis thaliana by Agrobacterium tumefaciens mediated. After Kan resistance screening, PCR identification of genomic DNA and real-time fluorescent quantitative PCR identification, 18 CFPS-M gene positive seedlings and 21 CFPS-2M gene positive seedlings were screened. The relative germination rate and root length of transgenic Arabidopsis thaliana seeds were higher than that of non-transgenic plants P0.05under various abiotic stresses. The transgenic Arabidopsis thaliana containing double copy of CFPS-M gene is more resistant to stress, especially under abiotic stress, such as drought, salinity, high temperature, low temperature and so on. The analysis of physiological and biochemical indexes of transgenic Arabidopsis thaliana seedlings showed that the malondialdehyde content of transgenic plants was significantly lower than that of wild type control. The contents of soluble protein, proline and other osmotic regulators were significantly higher than that of non-transgenic plants (P0.05N), and the activity of antioxidant enzyme system (SODPOD) CATwas significantly higher than that of wild type control (P0.05). It is suggested that CFPS-M and CFPS-2M genes decrease the content of MDA accumulated in plants and increase the content of osmotic regulatory protective substances in plants. The activity of antioxidant enzyme system to improve the ability of plants to resist abiotic stress.) the resistance of transgenic Arabidopsis thaliana plants with CFPS-2M gene is stronger than that of transgenic plants with CFPS-M gene. The results showed that increasing the copy number of exogenous stress resistance gene enhanced the expression of the stress resistance gene and made the plants more resistant to stress.
【學(xué)位授予單位】：西南科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2
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本文編號(hào)：1954147