樟樹LFY、UFO和FUL基因克隆及功能分析
發(fā)布時(shí)間：2018-10-26 13:03
【摘要】：香樟(Cinnamomum camphora)是樟目樟科樟屬的代表樹木,是一種常綠大喬木。成年樟樹枝葉繁茂,氣味芬香,是中國長江中下游地區(qū)常見園林樹種,廣泛用于園林綠化和市政道路綠化中。但是,樟樹的核果成熟后,會從樹上掉落下來,砸在行人、車輛以及地面上,給人們?nèi)粘Ｉ顜聿槐?同時(shí)還造成一定的污染。針對這個(gè)問題,本課題從植物花發(fā)育關(guān)鍵基因LFY、UFO、FUL出發(fā),分離克隆樟樹中這三個(gè)基因的同源基因,分別命名為Cc LFY、Cc UFO、Cc FUL,進(jìn)行基因功能分析,為以后獲得少花少果乃至無花無果的樟樹提供一定指導(dǎo)信息。本課題獲得Cc LFY、Cc UFO、Cc FUL序列后,構(gòu)建系統(tǒng)進(jìn)化樹,進(jìn)行基因序列分析和構(gòu)建系統(tǒng)進(jìn)化樹,然后進(jìn)行半定量表達(dá)分析,確定這三個(gè)基因功能。最后,構(gòu)建含目的基因的超表載體,進(jìn)行擬南芥的遺傳轉(zhuǎn)化,觀察分析轉(zhuǎn)基因擬南芥表型,進(jìn)行目的基因的功能鑒定。此外,本課題還進(jìn)行了酵母雙雜交實(shí)驗(yàn),分析Cc LFY、Cc UFO之間是否存在互作關(guān)系。本課題主要研究結(jié)果如下:(1)通過同源克隆技術(shù)、側(cè)翼擴(kuò)增技術(shù)和常規(guī)PCR,擴(kuò)增得到了Cc FUL基因全長序列;根據(jù)轉(zhuǎn)錄組數(shù)據(jù)、側(cè)翼擴(kuò)增技術(shù)和常規(guī)PCR,擴(kuò)增得到了Cc UFO基因全長序列;利用轉(zhuǎn)錄組數(shù)據(jù)和常規(guī)PCR,擴(kuò)增得到了Cc LFY基因全長序列。然后根據(jù)擴(kuò)增得到的序列,構(gòu)建系統(tǒng)進(jìn)化樹,進(jìn)行同源分析,結(jié)果表明這三個(gè)目的基因與雙子葉植物親緣關(guān)系較近。(2)采集樟樹不同部位和時(shí)期的組織,利用CTAB法提取RNA,進(jìn)行半定量表達(dá)分析,結(jié)果表明Cc LFY、Cc UFO、Cc FUL與樟樹花發(fā)育和果實(shí)形成有關(guān)。(3)利用p CAMBIA2300表達(dá)載體,通過傳統(tǒng)質(zhì)粒重組的方法,成功構(gòu)建了35S-Cc LFY、35S-Cc UFO、35S-Cc FUL等三個(gè)超表載體。然后將其電轉(zhuǎn)進(jìn)農(nóng)桿菌菌株GV1303中,最后通過農(nóng)桿菌介導(dǎo)的方法,進(jìn)行擬南芥的遺傳轉(zhuǎn)化,獲得了8株Cc LFY陽性轉(zhuǎn)基因擬南芥,11株Cc UFO陽性轉(zhuǎn)基因擬南芥以及6株Cc FUL陽性轉(zhuǎn)基因擬南芥。觀察陽性轉(zhuǎn)基因擬南芥的表型,發(fā)現(xiàn)Cc LFY、Cc UFO陽性轉(zhuǎn)基因擬南芥均出現(xiàn)了早花、側(cè)枝轉(zhuǎn)變?yōu)榛ǖ谋硇�。同時(shí)Cc LFY陽性轉(zhuǎn)基因擬南芥還出現(xiàn)分支少,植株底端側(cè)枝直接轉(zhuǎn)化成花的表型,Cc UFO陽性轉(zhuǎn)基因擬南芥還呈現(xiàn)出植株矮小的表型。此外,Cc FUL陽性轉(zhuǎn)基因擬南芥呈現(xiàn)晚花、果莢變短彎曲的表型。這些轉(zhuǎn)基因擬南芥表型有力證明了這三個(gè)基因與花發(fā)育和果實(shí)形成有關(guān)。(4)酵母雙雜交實(shí)驗(yàn)表明Cc LFY、Cc UFO之間的不存在互作關(guān)系。
[Abstract]:Cinnamomum camphora (Cinnamomum camphora) is the representative tree of Camphor family, and is a kind of large evergreen tree. The adult camphor tree is a common garden tree in the middle and lower reaches of the Yangtze River. It is widely used in landscape and municipal road greening. However, when the stone fruit of camphor tree ripens, it will fall down from the tree and crash on pedestrians, vehicles and the ground, which will bring inconvenience to people's daily life and cause certain pollution at the same time. In order to solve this problem, we isolated and cloned the homologous genes of the three genes in camphor tree from the key gene LFY,UFO,FUL of plant flower development, and named Cc LFY,Cc UFO,Cc FUL, for gene function analysis. It provides some guiding information for obtaining camphor trees with few flowers and few fruits or no flowers and no fruit. After the Cc LFY,Cc UFO,Cc FUL sequence was obtained, the phylogenetic tree was constructed, gene sequence analysis and phylogenetic tree were constructed, and semi-quantitative expression analysis was carried out to determine the function of the three genes. Finally, the supersurface vector containing the target gene was constructed, the genetic transformation of Arabidopsis thaliana was carried out, the phenotype of transgenic Arabidopsis was observed and analyzed, and the function of the target gene was identified. In addition, yeast two-hybrid experiments were carried out to analyze the interaction between Cc LFY,Cc UFO. The main results are as follows: (1) the full-length sequence of Cc FUL gene was obtained by homologous cloning, flanking amplification and conventional PCR, amplification. According to the transcriptional data, flanking amplification technique and conventional PCR, amplification, the full-length sequence of Cc UFO gene was obtained, and the full-length sequence of Cc LFY gene was obtained by using transcriptome data and conventional PCR, amplification. Then the phylogenetic tree was constructed according to the amplified sequence and the homology analysis was carried out. The results showed that the relationship between the three target genes and dicotyledonous plants was close. (2) the tissues of different parts and periods of camphor tree were collected and RNA, was extracted by CTAB method. The results of semi-quantitative expression analysis showed that Cc LFY,Cc UFO,Cc FUL was related to flower development and fruit formation of camphor tree. (3) 35S-Cc LFY,35S-Cc UFO, was successfully constructed by using p CAMBIA2300 expression vector and traditional plasmid recombination method. 35S-Cc FUL and other three supermeter carriers. At last, through Agrobacterium tumefaciens mediated transformation, 8 Cc LFY positive transgenic Arabidopsis thaliana strains were obtained. 11 Cc UFO positive transgenic Arabidopsis and 6 Cc FUL positive transgenic Arabidopsis thaliana. The phenotype of positive transgenic Arabidopsis thaliana was observed. It was found that all transgenic Arabidopsis thaliana with Cc LFY,Cc UFO positive appeared early flower and the lateral branch changed into flower phenotype. At the same time, Cc LFY positive transgenic Arabidopsis had fewer branches, and, Cc UFO positive transgenic Arabidopsis showed short phenotype. In addition, Cc FUL positive transgenic Arabidopsis thaliana showed the phenotype of late flower and short curving fruit pod. The phenotypes of these transgenic Arabidopsis thaliana showed that the three genes were related to flower development and fruit formation. (4) yeast two-hybrid experiments showed that there was no interaction between Cc LFY,Cc UFO.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S792.23
本文編號：2295805
[Abstract]:Cinnamomum camphora (Cinnamomum camphora) is the representative tree of Camphor family, and is a kind of large evergreen tree. The adult camphor tree is a common garden tree in the middle and lower reaches of the Yangtze River. It is widely used in landscape and municipal road greening. However, when the stone fruit of camphor tree ripens, it will fall down from the tree and crash on pedestrians, vehicles and the ground, which will bring inconvenience to people's daily life and cause certain pollution at the same time. In order to solve this problem, we isolated and cloned the homologous genes of the three genes in camphor tree from the key gene LFY,UFO,FUL of plant flower development, and named Cc LFY,Cc UFO,Cc FUL, for gene function analysis. It provides some guiding information for obtaining camphor trees with few flowers and few fruits or no flowers and no fruit. After the Cc LFY,Cc UFO,Cc FUL sequence was obtained, the phylogenetic tree was constructed, gene sequence analysis and phylogenetic tree were constructed, and semi-quantitative expression analysis was carried out to determine the function of the three genes. Finally, the supersurface vector containing the target gene was constructed, the genetic transformation of Arabidopsis thaliana was carried out, the phenotype of transgenic Arabidopsis was observed and analyzed, and the function of the target gene was identified. In addition, yeast two-hybrid experiments were carried out to analyze the interaction between Cc LFY,Cc UFO. The main results are as follows: (1) the full-length sequence of Cc FUL gene was obtained by homologous cloning, flanking amplification and conventional PCR, amplification. According to the transcriptional data, flanking amplification technique and conventional PCR, amplification, the full-length sequence of Cc UFO gene was obtained, and the full-length sequence of Cc LFY gene was obtained by using transcriptome data and conventional PCR, amplification. Then the phylogenetic tree was constructed according to the amplified sequence and the homology analysis was carried out. The results showed that the relationship between the three target genes and dicotyledonous plants was close. (2) the tissues of different parts and periods of camphor tree were collected and RNA, was extracted by CTAB method. The results of semi-quantitative expression analysis showed that Cc LFY,Cc UFO,Cc FUL was related to flower development and fruit formation of camphor tree. (3) 35S-Cc LFY,35S-Cc UFO, was successfully constructed by using p CAMBIA2300 expression vector and traditional plasmid recombination method. 35S-Cc FUL and other three supermeter carriers. At last, through Agrobacterium tumefaciens mediated transformation, 8 Cc LFY positive transgenic Arabidopsis thaliana strains were obtained. 11 Cc UFO positive transgenic Arabidopsis and 6 Cc FUL positive transgenic Arabidopsis thaliana. The phenotype of positive transgenic Arabidopsis thaliana was observed. It was found that all transgenic Arabidopsis thaliana with Cc LFY,Cc UFO positive appeared early flower and the lateral branch changed into flower phenotype. At the same time, Cc LFY positive transgenic Arabidopsis had fewer branches, and, Cc UFO positive transgenic Arabidopsis showed short phenotype. In addition, Cc FUL positive transgenic Arabidopsis thaliana showed the phenotype of late flower and short curving fruit pod. The phenotypes of these transgenic Arabidopsis thaliana showed that the three genes were related to flower development and fruit formation. (4) yeast two-hybrid experiments showed that there was no interaction between Cc LFY,Cc UFO.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S792.23
【相似文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 周琴;樟樹LFY、UFO和FUL基因克隆及功能分析[D];華中農(nóng)業(yè)大學(xué);2016年
，本文編號：2295805
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