玉米干旱胁迫应答NAC基因的克隆及功能分析

发布时间：2018-04-13 16:59

  本文选题：玉米 + NAC　； 参考：《西北农林科技大学》2017年硕士论文

【摘要】：植物在整个生长和发育过程中,时常会受到一些生物或者非生物胁迫的影响,比如病虫害、高温、干旱、盐碱以及激素等,它们严重危害了农作物的生长,影响其产量及品质。与此同时,为了适应并抵制各种逆境,植物在长期进化过程中,通过感知胁迫信号,调节与逆境胁迫相关的基因表达,形成了一系列有效地保护机制。NAC转录因子作为植物所特有的一种调节因子,在植物的形态建成、新陈代谢以及响应逆境胁迫中发挥重要的作用。本研究以玉米B73为材料,对8个玉米抗旱相关的候选ZmNAC基因进行克隆及生物信息学分析;运用实时定量PCR技术对它们在干旱胁迫下的表达模式进行分析。另外,选取表达上调的基因,以不同抗旱型玉米自交系为材料,对ZmNAC99及ZmNAC102进行核苷酸多态性及推断氨基酸序列分析,同时将其进行了拟南芥过表达及非生物胁迫抗逆性鉴定。主要研究结果如下:1.对克隆得到的8个ZmNAC基因(ZmNAC6,ZmNAC29,ZmNAC54,ZmNAC55,ZmNAC71,ZmNAC87,ZmNAC99及ZmNAC102)进行生物信息学分析,结果表明基因全长分别为1847bp、1105bp、2227bp、1988bp、2170bp、1135bp、1907bp和2152bp,与理论大小一致,其中除ZmNAC54含有5个外显子及4个内含子以外,其他7个候选ZmNAC基因均包含3个外显子和2个内含子;蛋白结构域的分析发现8个玉米候选抗旱相关ZmNAC的N-端都具有保守的NAM结构域;系统进化分析结果显示,8个玉米ZmNAC基因被分为两类;顺式元件分析揭示,除了包含CAAT-box和TATA-box等核心元件外,8个ZmNAC基因启动子中也存在非生物胁迫以及激素应答元件,如MBS、HSE及ABRE等。2.利用qRT-PCR技术对ZmNAC基因在不同干旱胁迫程度下的表达情况进行分析。结果表明:ZmNAC6基因在中等及严重干旱条件下均表达上调;而ZmNAC99和ZmNAC102基因仅在中等干旱或严重干旱的一种条件下表达上调;与之相反,ZmNAC55在中等及严重干旱条件下均表达下调。3.通过ClustalW对不同抗旱型玉米自交系来源的ZmNAC99和ZmNAC102的推断氨基酸序列分别进行比对,分析发现ZmNAC99和ZmNAC102在不同抗旱型自交系中存在氨基酸的差异,且在抗旱性相近的自交系中存在的差异明显少于抗旱性差异较大的自交系,推测基因结构的差异对玉米抗旱性可能产生一定影响。4.成功构建ZmNAC54、ZmNAC55、ZmNAC87、ZmNAC99和ZmNAC102的植物表达载体,通过蘸花法遗传转化拟南芥,得到T1代种子,同时通过含有100μg/m L的羧苄西林和30μg/m L的卡那霉素的1/2MS培养基进行筛选以及PCR检测,获得转基因ZmNAC99和ZmNAC102的T3代拟南芥种子。5.在控水法和300mmol/L甘露醇模拟的干旱胁迫下,与野生型对比,转ZmNAC99的拟南芥株系在幼苗期的根系相对较长,成株期叶片相对含水量较高,而失水率较低,这表明ZmNAC99在提高玉米抗旱能力方面可能具有一定的作用;45℃高温胁迫实验表明,ZmNAC102转基因植株在高温胁迫下呈现出比对照更强的耐受性;而在150mmo/L NaCl模拟的高盐胁迫以及2℃模拟的低温胁迫下,两个转基因拟南芥的植株与野生型拟南芥相比均没有明显的表型差别,说明ZmNAC99和ZmNAC102基因可能不能提高植物对高盐和低温的耐受性。
[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.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S513

【相似文献】

相关硕士学位论文 前2条

1 王中娜;亚洲棉NAC转录因子家族的生物信息学分析及相关NAC基因的克隆和功能研究[D];西南大学;2015年

2 熊结标;拟南芥转录因子CBF1及NAC基因的克隆与原核表达[D];南昌大学;2015年

，

本文编号：1745393