本文選題：玉米 + NAC　； 參考：《西北農(nóng)林科技大學》2017年碩士論文

【摘要】：植物在整個生長和發(fā)育過程中,時常會受到一些生物或者非生物脅迫的影響,比如病蟲害、高溫、干旱、鹽堿以及激素等,它們嚴重危害了農(nóng)作物的生長,影響其產(chǎn)量及品質(zhì)。與此同時,為了適應(yīng)并抵制各種逆境,植物在長期進化過程中,通過感知脅迫信號,調(diào)節(jié)與逆境脅迫相關(guān)的基因表達,形成了一系列有效地保護機制。NAC轉(zhuǎn)錄因子作為植物所特有的一種調(diào)節(jié)因子,在植物的形態(tài)建成、新陳代謝以及響應(yīng)逆境脅迫中發(fā)揮重要的作用。本研究以玉米B73為材料,對8個玉米抗旱相關(guān)的候選ZmNAC基因進行克隆及生物信息學分析;運用實時定量PCR技術(shù)對它們在干旱脅迫下的表達模式進行分析。另外,選取表達上調(diào)的基因,以不同抗旱型玉米自交系為材料,對ZmNAC99及ZmNAC102進行核苷酸多態(tài)性及推斷氨基酸序列分析,同時將其進行了擬南芥過表達及非生物脅迫抗逆性鑒定。主要研究結(jié)果如下:1.對克隆得到的8個ZmNAC基因(ZmNAC6,ZmNAC29,ZmNAC54,ZmNAC55,ZmNAC71,ZmNAC87,ZmNAC99及ZmNAC102)進行生物信息學分析,結(jié)果表明基因全長分別為1847bp、1105bp、2227bp、1988bp、2170bp、1135bp、1907bp和2152bp,與理論大小一致,其中除ZmNAC54含有5個外顯子及4個內(nèi)含子以外,其他7個候選ZmNAC基因均包含3個外顯子和2個內(nèi)含子;蛋白結(jié)構(gòu)域的分析發(fā)現(xiàn)8個玉米候選抗旱相關(guān)ZmNAC的N-端都具有保守的NAM結(jié)構(gòu)域;系統(tǒng)進化分析結(jié)果顯示,8個玉米ZmNAC基因被分為兩類;順式元件分析揭示,除了包含CAAT-box和TATA-box等核心元件外,8個ZmNAC基因啟動子中也存在非生物脅迫以及激素應(yīng)答元件,如MBS、HSE及ABRE等。2.利用qRT-PCR技術(shù)對ZmNAC基因在不同干旱脅迫程度下的表達情況進行分析。結(jié)果表明:ZmNAC6基因在中等及嚴重干旱條件下均表達上調(diào);而ZmNAC99和ZmNAC102基因僅在中等干旱或嚴重干旱的一種條件下表達上調(diào);與之相反,ZmNAC55在中等及嚴重干旱條件下均表達下調(diào)。3.通過ClustalW對不同抗旱型玉米自交系來源的ZmNAC99和ZmNAC102的推斷氨基酸序列分別進行比對,分析發(fā)現(xiàn)ZmNAC99和ZmNAC102在不同抗旱型自交系中存在氨基酸的差異,且在抗旱性相近的自交系中存在的差異明顯少于抗旱性差異較大的自交系,推測基因結(jié)構(gòu)的差異對玉米抗旱性可能產(chǎn)生一定影響。4.成功構(gòu)建ZmNAC54、ZmNAC55、ZmNAC87、ZmNAC99和ZmNAC102的植物表達載體,通過蘸花法遺傳轉(zhuǎn)化擬南芥,得到T1代種子,同時通過含有100μg/m L的羧芐西林和30μg/m L的卡那霉素的1/2MS培養(yǎng)基進行篩選以及PCR檢測,獲得轉(zhuǎn)基因ZmNAC99和ZmNAC102的T3代擬南芥種子。5.在控水法和300mmol/L甘露醇模擬的干旱脅迫下,與野生型對比,轉(zhuǎn)ZmNAC99的擬南芥株系在幼苗期的根系相對較長,成株期葉片相對含水量較高,而失水率較低,這表明ZmNAC99在提高玉米抗旱能力方面可能具有一定的作用;45℃高溫脅迫實驗表明,ZmNAC102轉(zhuǎn)基因植株在高溫脅迫下呈現(xiàn)出比對照更強的耐受性;而在150mmo/L NaCl模擬的高鹽脅迫以及2℃模擬的低溫脅迫下,兩個轉(zhuǎn)基因擬南芥的植株與野生型擬南芥相比均沒有明顯的表型差別,說明ZmNAC99和ZmNAC102基因可能不能提高植物對高鹽和低溫的耐受性。
[Abstract]:The plant in the whole growth and development process, will always be some biological or abiotic stress effects, such as pests, drought, salinity and temperature, hormone, which seriously endanger the growth of crops, affecting the yield and quality. At the same time, in order to adapt and resist all kinds of stress, plants in the long process of evolution through the perception, stress signal, regulation of stress-related gene expression, formed a series of effective protection mechanism of.NAC transcription factors as unique to plants are a regulatory factor in plant morphogenesis, and The new supersedes the old. play an important role in response to abiotic stress. In this study, corn B73 as material, analysis cloning and bioinformatics of 8 maize drought related candidate gene ZmNAC; using real-time quantitative PCR analysis of their expression patterns under drought stress were also, Selection of the up-regulated genes, with different drought resistant maize inbred lines, analyzed nucleotide polymorphisms and deduced amino acid sequences of ZmNAC99 and ZmNAC102, while its Arabidopsis overexpression and resistance to abiotic stress identification. The main results are as follows: 8 of the cloned ZmNAC gene 1. (ZmNAC6, ZmNAC29, ZmNAC54, ZmNAC55, ZmNAC71, ZmNAC87, ZmNAC99 and ZmNAC102) by bioinformatics analysis, results showed that the full-length genes were 1847bp, 1105bp, 2227bp, 1988bp, 2170bp, 1135bp, 1907bp and 2152bp, and the theory of the same size, except for ZmNAC54 which contains 5 exons and 4 introns, the other 7 the candidate ZmNAC gene contains 3 exons and 2 introns; analysis of protein domains found 8 candidate ZmNAC N- of maize drought resistance related end has a conserved NAM domain; phylogenetic analysis showed that 8 The maize ZmNAC gene was divided into two categories; cis element analysis revealed that in addition to containing CAAT-box and TATA-box core components, 8 ZmNAC gene promoter in abiotic stress and hormone response elements, there are sub such as MBS, HSE and ABRE.2. analysis of ZmNAC gene in different expression level under drought stress by using qRT-PCR technology. The results showed that ZmNAC6 genes were upregulated in moderate and severe drought conditions; while ZmNAC99 and ZmNAC102 were only in a moderate drought conditions or severe drought under the up regulation; on the contrary, the expression of ZmNAC55 was in moderate and severe drought conditions through down-regulation of.3. ClustalW deduced amino acid sequences of different drought resistant maize inbred lines from ZmNAC99 and ZmNAC102 respectively were compared, analysis showed that ZmNAC99 and ZmNAC102 are the differences of amino acid in different drought resistant and drought resistance in inbred lines. Differences between inbred lines in Their natures are much the same. significantly less than the drought resistance of different inbred lines were larger, speculated that the difference of gene structure may result in the successful construction of ZmNAC54, influence of.4. on Drought Resistance of maize ZmNAC55, ZmNAC87, ZmNAC99 and ZmNAC102 plant expression vector, genetic transformation of Arabidopsis by the floral dip method, T1 seeds, at the same time 1/2MS kanamycin medium screening and PCR detection card containing 100 g/m L carbenicillin and 30 g/m L, ZmNAC99 T3 and ZmNAC102 transgenic Arabidopsis seeds on behalf of.5. in water controlling and 300mmol/L mannitol simulation under drought stress, compared with the wild type, transgenic ZmNAC99 Arabidopsis plant relative in seedling root long, seedling leaf relative water content is high, and the rate of water loss was lower, suggesting that ZmNAC99 may play a role in improving the drought resistance ability; 45 degrees high temperature stress Forced experiments showed that ZmNAC102 transgenic plants under high temperature stress showed a stronger tolerance than the control; while high salt stress and NaCl 150mmo/L simulation in 2 C simulation under low temperature stress, two transgenic Arabidopsis plants compared with wild type Arabidopsis showed no obvious difference between phenotype and ZmNAC102 gene, indicating that ZmNAC99 may not improve plant tolerance to high salt and low temperature.

【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2;S513

【相似文獻】

相關(guān)碩士學位論文 前2條

1 王中娜;亞洲棉NAC轉(zhuǎn)錄因子家族的生物信息學分析及相關(guān)NAC基因的克隆和功能研究[D];西南大學;2015年

2 熊結(jié)標;擬南芥轉(zhuǎn)錄因子CBF1及NAC基因的克隆與原核表達[D];南昌大學;2015年

，

本文編號：1745393