香樟CBF类似基因的克隆及其在植物转基因抗寒育种中的潜在应用

发布时间：2018-03-18 04:31

本文选题：香樟　切入点：CBF/DREB1　出处：《南阳师范学院》2015年硕士论文　论文类型：学位论文

【摘要】：香樟(Cinnamomum camphora L.)属亚热带常绿阔叶树种,是一种优秀的园林绿化树种,在我国南方城市的园林绿化中有着较为广泛的应用。近年来,随着城市建设的快速发展,许多园林工作者尝试将香樟引种到中国北部,以丰富当地的常绿树种并改善冬天的园林景观。然而我国北方的冬天漫长而寒冷,严重限制了香樟的成功引种、推广和园林应用。加强栽培管理和选育抗寒性增强的香樟新品种是解决上述问题的关键,而通过改良香樟自身遗传因素提高对低温胁迫的耐受性是解决这一问题的最根本的方法。CBF(C-repeat binding factor)又称为DREB1(dehydration-responsive element binding factor 1),是一种重要的转录因子,在植物应对低温、干旱和高盐等逆境胁迫过程中起关键的作用。研究者们已经从许多植物中分离到编码CBF/DREB1转录因子的同源基因,不同来源的CBF/DREB1基因在序列和结构上有较高的相似性,超量表达该类基因可以增强转基因植物的一种或多种抗逆性,这一发现为通过转基因技术改良香樟抗寒性提供了新思路。本研究以前人对CBF/DERB1类转录因子的研究成果为基础,通过从香樟中分离编码低温诱导转录因子的CBF类似基因、探讨香樟CBF类似基因在不同逆境下的表达模式以及转香樟CBF类似基因烟草的抗逆性,对香樟CBF类似基因的功能进行初步探讨,为进一步培育转基因香樟奠定基础。本论文主要研究成果如下:1.首次从香樟中分离到4个Actin(肌动蛋白)基因片段,CcACTa、CcACTb、CcACTc和CcACTd(GenBank登录号:KM086736、KM086737、KM086738和KM086739)和1个EF1α(延伸因子)基因片段CcEF1α(登录号:KM086740),稳定性分析表明,CcACTc和CcEF1α基因在低温胁迫处理下表达量均相对稳定,可以作为香樟功能基因实时定量PCR分析的内参基因,且CcEF1α更为稳定。2.从香樟中成功分离4个CBF类似基因的cDNA序列全长,CcCBFa、CcCBFb、CcCBFc和CcCBFd(GenBank登录号:KJ958932、KJ958933、KP336741和KP336742),实时定量PCR分析结果表明,该4个基因可能在香樟抵御低温、干旱和高盐胁迫过程中有重要的作用,并且植物激素脱落酸(ABA)可能在其发挥作用时作为信号分子。3.通过基因重组技术构建了4个香樟CcCBF基因的植物表达载体CcCBF-p2300,并通过农杆菌介导的遗传转化方法获得了组成型表达CcCBFb基因的转基因烟草。抗逆性分析结果表明,转Cc CBFb基因烟草T1代幼苗的抗寒、抗旱以及抗盐胁迫能力明显高于野生型烟草。本课题是香樟抗寒分子育种的起始,还处于研究的起步阶段,但这是香樟抗寒分子育种不可或缺的重要环节,本研究找到了控制香樟抵御各种胁迫的重要转录因子,为实现通过转基因技术增强香樟抗寒性奠定了基础。
[Abstract]:Cinnamomum camphora L., a subtropical evergreen broad-leaved tree species, is an excellent landscaping tree species, which has been widely used in the landscape of southern cities in China. In recent years, along with the rapid development of urban construction, Cinnamomum camphora L. Many garden workers try to introduce camphor to northern China in order to enrich the local evergreen trees and improve the landscape in winter. However, the long and cold winter in northern China seriously limits the successful introduction of Cinnamomum camphora. The key to solve the above problems is to strengthen cultivation and management and to select new camphor varieties with enhanced cold resistance. The most fundamental method to solve this problem is to improve the tolerance to low temperature stress by improving the genetic factors of Cinnamomum camphora. The CBFC-repeat binding factor, also called DREB1(dehydration-responsive element binding factor 1, is an important transcription factor, which can be used to deal with low temperature in plants. Drought and high salt stress play a key role in the process of stress. Researchers have isolated homologous genes encoding CBF/DREB1 transcription factors from many plants. CBF/DREB1 genes from different sources have high similarity in sequence and structure. Overexpression of these genes can enhance the resistance of transgenic plants to one or more kinds of stresses, which provides a new way to improve the cold resistance of Cinnamomum camphora by transgenic technology. CBF analogous genes encoding low temperature induced transcription factors were isolated from Cinnamomum camphora to investigate the expression patterns of CBF analogous genes under different stresses and the resistance of transgenic tobacco plants with similar CBF genes. The function of analogous gene of CBF in Cinnamomum camphora was studied. The main results of this thesis are as follows: 1. For the first time, four actin (actin) gene fragments were isolated from Cinnamomum camphora, CcACTbCCTc and CcACTd(GenBank accession numbers: KM086736, KM086737, KM086738 and KM086739) and one EF1 伪 (elongation factor) gene fragment, CcACTc and CcACTd(GenBank accession numbers: KM086736, KM086737 and KM086739. The stability analysis of CcEF1 伪 (accession number: KM086740) showed that the expression of CcACTc and CcEF1 伪 genes were relatively stable under low temperature stress. It can be used as an internal reference gene for real-time quantitative PCR analysis of Cinnamomum camphora functional gene, and CcEF1 伪 is more stable. 2. Four full-length cDNA sequences of similar CBF genes were successfully isolated from Cinnamomum camphora. The results of real-time quantitative PCR analysis showed that: KJ958932, KJ9589332, KJ958933P336741 and KP336742 were successfully isolated from Cinnamomum camphora, and four full-length cDNA sequences of similar CBF genes were successfully isolated from Cinnamomum camphora. These four genes may play an important role in the resistance of Cinnamomum camphora to low temperature, drought and high salt stress. And the plant hormone abscisic acid (Aba) may act as signal molecule. 3. Four plant expression vectors of CcCBF gene of Cinnamomum camphora CcCBF-p2300 were constructed by gene recombination technique, and obtained by Agrobacterium tumefaciens mediated genetic transformation. Constitutive expression of CcCBFb gene in transgenic tobacco. The ability of resistance to cold, drought and salt stress in T1 generation of transgenic tobacco with C c CBFb gene was obviously higher than that of wild type tobacco. This subject is the beginning of molecular breeding of camphor camphor resistance to cold, which is still in the initial stage of study. However, this is an indispensable link in the molecular breeding of camphora camphora. In this study, an important transcription factor was found to control camphor resistance to various stresses, which laid a foundation for enhancing the cold resistance of Cinnamomum camphora through transgenic technology.
【学位授予单位】：南阳师范学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q943.2;S792.23

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