【作者】 张丽；

【导师】 韩兆雪；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2017， 硕士

【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。更多还原

【Abstract】 In the process of growth and development,plants are often affected by biotic or abiotic stresses,such as disease and insect pests,high temperature,drought,salinity and hormones,they seriously affect the growth,yield and quality of crops.Meanwhile,in order to adapt to and resist all kinds of adversity,a series of effective protection mechanisms have been formed by sensing the stress signals and regulating the expression of genes involved in stress response in the long term evolution of plants.NAC transcription factor,as a unique regulatory factor,plays an important role in plant morphogenesis,metabolism and response to stress.In this study,8 NAC candidate genes related to drought resistance in Maize were cloned from maize inbred line B73 and carried on the bioinformatics analysis of them;The expression patterns of them under drought stress were analyzed by real-time quantitative PCR.In addition,up-regulated genes were used to analyze the nucleotide polymorphism and deduced amino acid sequence of maize inbred lines with different drought resistance;at the same time,The overexpression and abiotic stress tolerance of Arabidopsis thaliana were also identified.Here are the main results:1.Bioinformatics analysis of 8 ZmNAC genes(ZmNAC6,ZmNAC29,ZmNAC54,ZmNAC55,ZmNAC71,ZmNAC87,ZmNAC99,and ZmNAC102)were cloned,The results showed that the full-length of genes were 1847 bp,1105bp,2227 bp,1988bp,2170 bp,1135bp,1907 bp and 2152 bp respectively,consistent with the theoretical size,In addition to ZmNAC54 containing 4 introns and 5 exons,the other 7 candidate ZmNAC genes all contained 2 introns and 3 exons.Protein domain analysis revealed that the N-terminal of the drought resistance of 8 maize candidate ZmNAC genes had a conserved NAM domain;Phylogenetic analysis showed that the 8 maize ZmNAC genes were divided into two categories;The cis-element revealed that in addition to containing core promoter elements such as CAAT-box and TATA-box,abiotic stress and hormone responsive elements are also present in the promoters of the 8 ZmNAC genes,such as MBS,HSE and ABRE,etc.2.The expression of ZmNAC gene under different drought stress was analyzed by qRT-PCR technique.The results showed that ZmNAC6 gene was up-regulated in moderate and severe drought conditions;However,ZmNAC99 and ZmNAC102 genes were up-regulated only under the condition of moderate drought and severe drought.On the contrary,ZmNAC55 was down regulated under moderate drought and severe drought.3.The deduced amino acid sequences of ZmNAC99 and ZmNAC102 from different drought resistant maize inbred lines were compared by ClustalW.The results showed that ZmNAC99 and ZmNAC102 had different amino acid differences in different drought resistant inbred lines,and the difference between the inbred lines with similar drought resistance was significantly less than that of drought resistant inbred lines,and we speculated that the difference of genetic structure may have an effect on the drought resistance of maize.4.The plant expression vectors of ZmNAC54,ZmNAC55,ZmNAC87,ZmNAC99 and ZmNAC102 were successfully constructed,and transformed into Arabidopsis thaliana by dipping flower method to obtain T1 generation seeds,At the same time the T1 transgenic Arabidopsis seed of ZmNAC99 and ZmNAC102 were screened by 1/2MS medium containing 100 μg/mL Carbenicillin and 30 μg/mL Kanamycin and PCR detected,until the T3 generation transgenic Arabidopsis seed were harvested.5.Under the drought stress simulated by water control and 300mmol/L mannitol,Compared with wild type,the roots of ZmNAC99 transgenic Arabidopsis plants were relatively longer and the relative water content of leaves was higher while the rate of water loss was low in adult plant stage.This suggests that ZmNAC99 may play a role in improving drought resistance in Maize.45℃high temperature stress experiment showed that ZmNAC102 transgenic plants showed better tolerance than controls under high temperature stress.However,under the high salinity stress of 150mmo/L NaCl and the low temperature stress of 2 ℃,there was no obvious phenotypic difference between two transgenic Arabidopsis plants and wild-type Arabidopsis,the results showed that ZmNAC99 and ZmNAC102 could not improve the ability of salt tolerance and cold tolerance.更多还原