【摘要】：茶樹(Camellia sinensis(L.)O.Kuntze)為多年生常綠木本植物,是我國重要的葉用經(jīng)濟植物。茶樹全年的生長由營養(yǎng)生長和生殖生長組成,開花屬茶樹的生殖生長,是形成茶果的必要階段。茶樹開花結(jié)果會爭奪茶葉養(yǎng)分,致使茶葉產(chǎn)量下降、質(zhì)量降低。因此,控制茶樹的生殖生長、促進營養(yǎng)生長是茶樹產(chǎn)量和品質(zhì)提高的關(guān)鍵。如何合理調(diào)控茶樹的生殖生長和營養(yǎng)生長,從根本上提高茶樹產(chǎn)量和品質(zhì),是茶樹研究工作者亟待解決的問題。依據(jù)現(xiàn)代植物花發(fā)育"ABCDE模型”等研究發(fā)現(xiàn),B類和C類基因?qū)ǖ男詣e分化和功能發(fā)育具有重要作用。因此有必要開展茶樹B類MADS-box基因的克隆及其參與生長發(fā)育的分子機理研究,闡明其在轉(zhuǎn)錄和分子水平對茶樹花發(fā)育的調(diào)控機制,為今后利用生物技術(shù)手段控制茶樹的生殖生長提供重要的理論依據(jù)。本研究以陜西省秦巴山區(qū)代表性茶樹種質(zhì)‘紫陽櫧葉種’((Camellia sinesis cv. Ziyangzhong)為材料,通過開放閱讀框法和RACE技術(shù)相結(jié)合的方法獲得茶樹B類MADS-box同源基因CsTM6的cDNA序列全長,并進行了生物信息學(xué)預(yù)測和系統(tǒng)進化分析；另外,通過擬南芥(Arabidopsis thaliana)異位表達(dá)技術(shù)對CsTM6和CsDEF基因進行了功能分析,還利用實時熒光定量PCR、亞細(xì)胞定位分析、雙分子熒光互補技術(shù)(BiFC)、轉(zhuǎn)錄激活活性分析和酵母雙雜交技術(shù)初步解析了這兩個基因在茶樹花發(fā)育過程中的作用機理。主要結(jié)論如下：1.結(jié)合RT-PCR和RACE技術(shù)以‘紫陽櫧葉種’已分化花芽為材料,克隆到一個編碼框為630bp的cDNA序列。經(jīng)BLASTN比對分析和生物信息學(xué)預(yù)測,這個茶樹基因與GenBank數(shù)據(jù)庫中的山茶(Camellia japonica)、油茶(Camellia oleifera)等多個物種的TM6同源基因相似,且都具有保守的MADS域、K域和paleoAP3基序。故命名為CsTM6,GenBank登錄號為KR869823。2.對CsTM6編碼蛋白進行保守結(jié)構(gòu)域分析、亞細(xì)胞定位預(yù)測、疏水性／親水性分析、二級結(jié)構(gòu)預(yù)測、同源建模三維結(jié)構(gòu)預(yù)測和同源性比對分析表明：CsTM6屬于花發(fā)育B類基因,編碼MIKC類MADS-box轉(zhuǎn)錄因子；系統(tǒng)進化分析顯示茶樹CsTM6基因被準(zhǔn)確歸類到B類TM6進化分支,茶樹CsTM6基因與進化樹中B類基因AP3/DEF進化系18個物種的親緣性劃分與植物傳統(tǒng)分類相一致,CsTM6基因與山茶花的CjTM6和油茶CoTM6相似性最高。3.實時熒光定量PCR結(jié)果顯示,CsTM6和CsDEF在茶樹六類組織和器官中都有不同程度的表達(dá),但兩者的表達(dá)模式不同。CsTM6在花瓣、雄蕊和果實中的表達(dá)量高,在心皮的表達(dá)量次之,在萼片中的表達(dá)量較花芽和芽葉中的表達(dá)量略高；CsDEF在不同組織中都有表達(dá),但表達(dá)的豐度不同,其在花瓣中的表達(dá)量高,在萼片、雄蕊、果實和花芽的表達(dá)量次之,在心皮和芽葉中的表達(dá)量最低。表現(xiàn)出茶樹CsTM6和CsDEF基因表達(dá)模式明顯的種屬特異性。作為花器官特征基因,CsTM6和CsDEF在第二、三輪花器官中高表達(dá)共同調(diào)控花瓣和雄蕊發(fā)育,另外,這2個基因在果實中的高表達(dá)還暗示它們可能共同參與茶樹果實的生長發(fā)育。4.擬南芥中的異位表達(dá)結(jié)果顯示,35S::CsTM6和35S::CsDEF轉(zhuǎn)基因擬南芥植株表型差異較大,CsTM6在擬南芥中的異位表達(dá)表現(xiàn)為花瓣缺失,并嚴(yán)重影響花瓣的發(fā)育；CsDEF基因在擬南芥中的異位表達(dá)引起花瓣不同程度的卷曲。5.亞細(xì)胞定位分析和轉(zhuǎn)錄激活活性實驗發(fā)現(xiàn),CsTM6和CsDEF蛋白是具備核定位能力,但無轉(zhuǎn)錄激活活性的轉(zhuǎn)錄因子；酵母雙雜交實驗中兩者都與CsGLO1蛋白相互作用形成異源二聚體,且CsDEF還可與CsAG相互作用形成異源二聚體；BiFC實驗中CsTM6和CsDEF都可與CsGLO1、CsGLO2和CsAG相互作用形成異源二聚體。兩種實驗結(jié)果蛋白互作模式的差異,可能由于CsTM6和CsDEF基因編碼序列間存在差異,并且預(yù)示著可能需要通過與其他轉(zhuǎn)錄因子結(jié)合獲取轉(zhuǎn)錄調(diào)控活性參與花發(fā)育調(diào)控。
[Abstract]:Tea tree ( Camellia sinensis ( L . ) O . Kunyu ) is a perennial evergreen woody plant . It is an important leaf economic plant in China . The growth of tea tree is the key to the development of tea tree . Ziyangzhong was used as a material to obtain the full length of cDNA sequence of MADS - box homologous gene CsTM6 of tea tree by open reading frame method and RACE technology , and bioinformatics prediction and systematic evolution analysis were carried out .
The roles of CsTM6 and CsDEF genes in the development of Camellia japonica were analyzed by means of real - time fluorescence quantitative PCR , subcell localization analysis , bimolecular fluorescence complementary technology ( BiFC ) , transcriptional activation analysis and yeast two - hybrid technique .
The phylogenetic analysis showed that CsTM6 gene was accurately classified into B - type TM6 , and the relationship between CsTM6 gene and C _ jTM6 and oil - tea CoTM6 was highest . The expression of CsTM6 gene was higher in petals , stamens and fruits . The expression of CsTM6 in sepals , stamens and fruit was higher than that in flower buds and buds .
The expression of CsTM6 and CsDEF in flower petals is the lowest . The expression of CsTM6 and CsDEF in flower petals is the lowest . The expression of CsTM6 and CsDEF in flower petals is the lowest . In addition , the high expression of CsTM6 and CsDEF in the second and third round flower organs shows that they may participate in the development of flower organs .
The ectopic expression of CsDEF gene in Arabidopsis resulted in different degrees of curl . 5 . The cell location analysis and transcriptional activation assay showed that CsTM6 and CsDEF protein were transcription factors possessing nuclear localization ability , but no transcriptional activation activity .
In the yeast two - hybrid experiment , both of them interact with CsGLO1 protein to form heterodimer , and CsDEF can also interact with CsAG to form heterodimer ;
CsTM6 and CsDEF can interact with CsGLO1 , CsGLO2 and CsAG to form heterodimers . The difference between the two experimental results suggests that there is a difference between CsTM6 and CsDEF coding sequences , and it may be necessary to participate in the development and regulation of flower development by combining with other transcription factors .
【學(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S571.1;Q943.2

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1 唐紅;茶樹MADS-box家族B類基因CsTM6的克隆及其與CsDEF的功能研究[D];陜西師范大學(xué);2016年

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