本文選題：香樟　切入點：CBF/DREB1　出處：《南陽師范學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：香樟(Cinnamomum camphora L.)屬亞熱帶常綠闊葉樹種,是一種優(yōu)秀的園林綠化樹種,在我國南方城市的園林綠化中有著較為廣泛的應(yīng)用。近年來,隨著城市建設(shè)的快速發(fā)展,許多園林工作者嘗試將香樟引種到中國北部,以豐富當?shù)氐某＞G樹種并改善冬天的園林景觀。然而我國北方的冬天漫長而寒冷,嚴重限制了香樟的成功引種、推廣和園林應(yīng)用。加強栽培管理和選育抗寒性增強的香樟新品種是解決上述問題的關(guān)鍵,而通過改良香樟自身遺傳因素提高對低溫脅迫的耐受性是解決這一問題的最根本的方法。CBF(C-repeat binding factor)又稱為DREB1(dehydration-responsive element binding factor 1),是一種重要的轉(zhuǎn)錄因子,在植物應(yīng)對低溫、干旱和高鹽等逆境脅迫過程中起關(guān)鍵的作用。研究者們已經(jīng)從許多植物中分離到編碼CBF/DREB1轉(zhuǎn)錄因子的同源基因,不同來源的CBF/DREB1基因在序列和結(jié)構(gòu)上有較高的相似性,超量表達該類基因可以增強轉(zhuǎn)基因植物的一種或多種抗逆性,這一發(fā)現(xiàn)為通過轉(zhuǎn)基因技術(shù)改良香樟抗寒性提供了新思路。本研究以前人對CBF/DERB1類轉(zhuǎn)錄因子的研究成果為基礎(chǔ),通過從香樟中分離編碼低溫誘導(dǎo)轉(zhuǎn)錄因子的CBF類似基因、探討香樟CBF類似基因在不同逆境下的表達模式以及轉(zhuǎn)香樟CBF類似基因煙草的抗逆性,對香樟CBF類似基因的功能進行初步探討,為進一步培育轉(zhuǎn)基因香樟奠定基礎(chǔ)。本論文主要研究成果如下:1.首次從香樟中分離到4個Actin(肌動蛋白)基因片段,CcACTa、CcACTb、CcACTc和CcACTd(GenBank登錄號:KM086736、KM086737、KM086738和KM086739)和1個EF1α(延伸因子)基因片段CcEF1α(登錄號:KM086740),穩(wěn)定性分析表明,CcACTc和CcEF1α基因在低溫脅迫處理下表達量均相對穩(wěn)定,可以作為香樟功能基因?qū)崟r定量PCR分析的內(nèi)參基因,且CcEF1α更為穩(wěn)定。2.從香樟中成功分離4個CBF類似基因的cDNA序列全長,CcCBFa、CcCBFb、CcCBFc和CcCBFd(GenBank登錄號:KJ958932、KJ958933、KP336741和KP336742),實時定量PCR分析結(jié)果表明,該4個基因可能在香樟抵御低溫、干旱和高鹽脅迫過程中有重要的作用,并且植物激素脫落酸(ABA)可能在其發(fā)揮作用時作為信號分子。3.通過基因重組技術(shù)構(gòu)建了4個香樟CcCBF基因的植物表達載體CcCBF-p2300,并通過農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化方法獲得了組成型表達CcCBFb基因的轉(zhuǎn)基因煙草。抗逆性分析結(jié)果表明,轉(zhuǎn)Cc CBFb基因煙草T1代幼苗的抗寒、抗旱以及抗鹽脅迫能力明顯高于野生型煙草。本課題是香樟抗寒分子育種的起始,還處于研究的起步階段,但這是香樟抗寒分子育種不可或缺的重要環(huán)節(jié),本研究找到了控制香樟抵御各種脅迫的重要轉(zhuǎn)錄因子,為實現(xiàn)通過轉(zhuǎn)基因技術(shù)增強香樟抗寒性奠定了基礎(chǔ)。
[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 偽 (elongation factor) gene fragment, CcACTc and CcACTd(GenBank accession numbers: KM086736, KM086737 and KM086739. The stability analysis of CcEF1 偽 (accession number: KM086740) showed that the expression of CcACTc and CcEF1 偽 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 偽 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.
【學(xué)位授予單位】：南陽師范學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：Q943.2;S792.23

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相關(guān)期刊論文 前1條

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