玉米ZmMYBR58转录因子基因的克隆及功能分析

发布时间：2018-04-10 00:34

本文选题：玉米　切入点：非生物胁迫　出处：《甘肃农业大学》2016年硕士论文

【摘要】：非生物胁迫是致使世界范围内产生粮食危机的主要因素之一,全球每年因非生物胁迫至少能导致农作物减产50%。玉米是重要的粮食、饲料及工业原料,其生育期极易受干旱、高盐、低温等多种胁迫因子的干扰,是逆境敏感型植物。在面临着全球总人数不断上升而可种植面积不断减少的窘境下,探索植物对非生物胁迫的应答原理以及挖掘能够参与调节植物生长抗逆过程的基因是非常必要的。MYB是植物中最为普遍存在的转录因子家族,几乎参与调控了植物的全部生命活动,在植物抵抗非生物胁迫的过程当中也发挥出着重要的调控作用。本研究从玉米自交系F83中克隆得到一个MYB转录基因,并通过对该基因进行生物学信息分析、实时荧光定量PCR以及转基因植株的抗性分析,初步研究了Zm MYBR58基因的基本性质及其生物学功能。研究结果如下:1利用RT-PCR法克隆,从玉米自交系F83中得到Zm MYBR58基因,该基因编码的蛋白共包括433个氨基酸,包含了一个全长1302 bp的开放阅读框;预测蛋白理论分子量为46.0966 KD,理论等电点为9.52,理论半衰期为30 h,属于不稳定蛋白,亲水性氨基酸多于疏水性氨基酸,是一种亲水性蛋白;同源性比较结果显示该基因分别与高粱、谷子、小麦、水稻等作物的MYB蛋白具有一定的相似性。2蛋白分析显示Zm MYBR58基因编码的蛋白序列仅具有一个保守的SANT结构,能形成3个α螺旋结构,帮助折叠成正确的蛋白结构,属于MYB-related家族。3根据Zm MYBR58基因上游序列搜索其顺式作用元件发现存在光诱导、激素诱导、干旱诱导、低温诱导、组织特异表达等一系列相关元件,预测Zm MYBR58基因可能能够被干旱、低温、激素等胁迫因子诱导,从而参与到植物对逆境的响应过程中。4表达分析结果显示Zm MYBR58基因在玉米各检测组织中均有表达,其中在玉米胚芽中的表达量显著高于其他组织,具有组织特异性;Zm MYBR58基因的表达模式受干旱、高盐、ABA、低温以及高温的诱导,且其表达模式受不同诱导的响应程度不同,说明该基因可能参与调控着植物抗逆机制中的激素/逆境信号传递通路。5成功构建了CPB-Zm MYBR58重组载体,利用农杆菌介导法转化拟南芥,经过除草剂筛选和PCR鉴定得到Zm MYBR58转基因拟南芥的抗性植株,干旱处理下转基因抗性植株表现出较野生型拟南芥更高的耐旱性。以上研究结果均表明Zm MYBR58基因能够参与调节植物的非生物胁迫耐受性,本研究初步分析了Zm MYBR58基因的结构和功能,为进一步分析该基因参与的植物胁迫响应调节机制奠定了理论依据,也为今后利用基因工程相关技术培育玉米抗逆品种提供了一个优良的候选基因。
[Abstract]:Abiotic stress is one of the main factors leading to the food crisis in the world.Maize is an important food, feed and industrial raw material. It is susceptible to drought, high salt, low temperature and other stress factors in its growth period.Faced with a growing global population and declining plantable areas,It is necessary to explore the principles of plant response to abiotic stress and to dig up genes that can regulate plant growth and stress resistance. MYB is the most common transcription factor family in plants.It plays an important role in the process of plant resistance to abiotic stress.In this study, a MYB transcription gene was cloned from maize inbred line F83 and analyzed by biological information, real-time quantitative PCR and resistance analysis of transgenic plants.The basic properties and biological functions of Zm MYBR58 gene were preliminarily studied.The results were as follows: 1. The Zm MYBR58 gene was cloned by RT-PCR from maize inbred line F83. The protein encoded by the gene contained 433 amino acids and contained an open reading frame with a length of 1302 BP.The theoretical molecular weight of the predicted protein is 46.0966 KD, the theoretical isoelectric point is 9.52, the theoretical half-life is 30 h, the predicted protein belongs to unstable protein, hydrophilic amino acid is more than hydrophobic amino acid, and it is a kind of hydrophilic protein.The MYB protein of millet, wheat, rice and other crops showed that the MYB protein sequence encoded by Zm MYBR58 gene had only one conserved SANT structure, and could form three 伪 helical structures, helping to fold into the correct protein structure.According to the upstream sequence of Zm MYBR58 gene, a series of related elements, such as light induction, hormone induction, drought induction, hypothermia induction and tissue specific expression, were found in MYB-related family. It was predicted that Zm MYBR58 gene might be drought induced.The results showed that Zm MYBR58 gene was expressed in all the tested tissues of maize, and the expression level of Zm MYBR58 in maize germ was significantly higher than that in other tissues.The expression pattern of Zm MYBR58 gene with tissue specificity was induced by drought, high salinity, low temperature and high temperature, and its expression pattern was different in response to different induction.These results suggest that the gene may be involved in regulating the hormone / stress signaling pathway in plant stress resistance mechanism. 5. The recombinant vector of CPB-Zm MYBR58 was successfully constructed and transformed into Arabidopsis thaliana by Agrobacterium tumefaciens.The resistant plants of transgenic Arabidopsis thaliana (Arabidopsis thaliana) with Zm MYBR58 gene were obtained by herbicide screening and PCR identification. The transgenic plants showed higher drought resistance than wild type Arabidopsis thaliana under drought treatment.All the results indicated that Zm MYBR58 gene was involved in the regulation of abiotic stress tolerance in plants. The structure and function of Zm MYBR58 gene were preliminarily analyzed in this study.It provides a theoretical basis for further analysis of the regulation mechanism of plant stress response in which the gene is involved, and also provides a good candidate gene for the cultivation of stress resistant maize varieties by genetic engineering techniques in the future.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S513

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