茶树MADS-box家族B类基因CsTM6的克隆及其与CsDEF的功能研究
发布时间：2018-07-16 23:38
【摘要】：茶树(Camellia sinensis(L.)O.Kuntze)为多年生常绿木本植物,是我国重要的叶用经济植物。茶树全年的生长由营养生长和生殖生长组成,开花属茶树的生殖生长,是形成茶果的必要阶段。茶树开花结果会争夺茶叶养分,致使茶叶产量下降、质量降低。因此,控制茶树的生殖生长、促进营养生长是茶树产量和品质提高的关键。如何合理调控茶树的生殖生长和营养生长,从根本上提高茶树产量和品质,是茶树研究工作者亟待解决的问题。依据现代植物花发育"ABCDE模型”等研究发现,B类和C类基因对花的性别分化和功能发育具有重要作用。因此有必要开展茶树B类MADS-box基因的克隆及其参与生长发育的分子机理研究,阐明其在转录和分子水平对茶树花发育的调控机制,为今后利用生物技术手段控制茶树的生殖生长提供重要的理论依据。本研究以陕西省秦巴山区代表性茶树种质‘紫阳槠叶种’((Camellia sinesis cv. Ziyangzhong)为材料,通过开放阅读框法和RACE技术相结合的方法获得茶树B类MADS-box同源基因CsTM6的cDNA序列全长,并进行了生物信息学预测和系统进化分析；另外,通过拟南芥(Arabidopsis thaliana)异位表达技术对CsTM6和CsDEF基因进行了功能分析,还利用实时荧光定量PCR、亚细胞定位分析、双分子荧光互补技术(BiFC)、转录激活活性分析和酵母双杂交技术初步解析了这两个基因在茶树花发育过程中的作用机理。主要结论如下：1.结合RT-PCR和RACE技术以‘紫阳槠叶种’已分化花芽为材料,克隆到一个编码框为630bp的cDNA序列。经BLASTN比对分析和生物信息学预测,这个茶树基因与GenBank数据库中的山茶(Camellia japonica)、油茶(Camellia oleifera)等多个物种的TM6同源基因相似,且都具有保守的MADS域、K域和paleoAP3基序。故命名为CsTM6,GenBank登录号为KR869823。2.对CsTM6编码蛋白进行保守结构域分析、亚细胞定位预测、疏水性／亲水性分析、二级结构预测、同源建模三维结构预测和同源性比对分析表明：CsTM6属于花发育B类基因,编码MIKC类MADS-box转录因子；系统进化分析显示茶树CsTM6基因被准确归类到B类TM6进化分支,茶树CsTM6基因与进化树中B类基因AP3/DEF进化系18个物种的亲缘性划分与植物传统分类相一致,CsTM6基因与山茶花的CjTM6和油茶CoTM6相似性最高。3.实时荧光定量PCR结果显示,CsTM6和CsDEF在茶树六类组织和器官中都有不同程度的表达,但两者的表达模式不同。CsTM6在花瓣、雄蕊和果实中的表达量高,在心皮的表达量次之,在萼片中的表达量较花芽和芽叶中的表达量略高；CsDEF在不同组织中都有表达,但表达的丰度不同,其在花瓣中的表达量高,在萼片、雄蕊、果实和花芽的表达量次之,在心皮和芽叶中的表达量最低。表现出茶树CsTM6和CsDEF基因表达模式明显的种属特异性。作为花器官特征基因,CsTM6和CsDEF在第二、三轮花器官中高表达共同调控花瓣和雄蕊发育,另外,这2个基因在果实中的高表达还暗示它们可能共同参与茶树果实的生长发育。4.拟南芥中的异位表达结果显示,35S::CsTM6和35S::CsDEF转基因拟南芥植株表型差异较大,CsTM6在拟南芥中的异位表达表现为花瓣缺失,并严重影响花瓣的发育；CsDEF基因在拟南芥中的异位表达引起花瓣不同程度的卷曲。5.亚细胞定位分析和转录激活活性实验发现,CsTM6和CsDEF蛋白是具备核定位能力,但无转录激活活性的转录因子；酵母双杂交实验中两者都与CsGLO1蛋白相互作用形成异源二聚体,且CsDEF还可与CsAG相互作用形成异源二聚体；BiFC实验中CsTM6和CsDEF都可与CsGLO1、CsGLO2和CsAG相互作用形成异源二聚体。两种实验结果蛋白互作模式的差异,可能由于CsTM6和CsDEF基因编码序列间存在差异,并且预示着可能需要通过与其他转录因子结合获取转录调控活性参与花发育调控。
[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 .
【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S571.1;Q943.2
本文编号：2128067
[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 .
【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S571.1;Q943.2
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1 唐红;茶树MADS-box家族B类基因CsTM6的克隆及其与CsDEF的功能研究[D];陕西师范大学;2016年
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