【作者】 肖政；

【导师】 李纪元；

【作者基本信息】 中国林业科学研究院 ， 园林植物与观赏园艺， 2014， 博士

【摘要】 GA20-氧化酶和GA2-氧化酶是赤霉素合成代谢途径中的两个关键酶，都由多基因家族编码，参与调控植物内源活性GA水平。目前模式植物中GA20-氧化酶和GA2-氧化酶的功能研究较多，但在山茶属植物中GA20-氧化酶和GA2-氧化酶基因的功能及调控株型等研究还未见报道。本研究采用同源克隆方法从荔波连蕊茶(Camellia lipoensis Chang et Xu)中分离出5个与赤霉素合成代谢相关基因的cDNA全长：GA20-氧化酶基因2个和GA2-氧化酶基因3个，分别命名为ClGA20ox1、ClGA20ox2、ClGA2ox1、ClGA2ox2和ClGA2ox3。采用生物信息学软件分析基因序列，表明这些基因是所克隆的目的基因。实验获得的GA20-氧化酶和GA2-氧化酶基因在大肠杆菌E.Coli BL21中经IPTG诱导能表达出相应的蛋白，表明克隆的GA20-氧化酶和GA2-氧化酶基因具有在真核生物中编码蛋白质的能力。荧光定量PCR分析GA20-氧化酶和GA2-氧化酶在在荔波连蕊茶不同器官及发育的不同时期的表达模式。ClGA20ox1和ClGA20ox2基因在当年抽生的幼嫩茎中的表达量均较低，在茎和根中的表达量均较高。其中ClGA20ox1基因在种子的表达量最高，在嫩叶中的表达量最低。ClGA20ox2基因在茎中的表达量最高，在当年新抽生的幼嫩茎尖表达量最低。表明ClGA20ox1基因可能主要参与植物种子和根的生长发育调控，ClGA20ox2基因可能主要参与植物茎和叶的生长发育调控。ClGA2ox1、ClGA2ox2和ClGA2ox3基因在成熟叶片中都有较高表达，在种子和嫩叶中的表达量都较低。其中ClGA2ox1基因在茎中的表达量最高，在嫩叶中的表达量最低。ClGA2ox2基因在荔波连蕊茶的成熟叶片中表达量最高，在种子中的表达量最低。ClGA2ox3基因在根中的表达量最高，在嫩叶中的表达量最低。表明ClGA2ox1基因可能主要参与植物茎和叶的生长发育；ClGA2ox2基因可能主要参与植物煮熟叶片的衰老代谢；ClGA2ox3基因可能主要参与根的生长发育。利用pCAMBIA1300表达载体分别构建了ClGA20ox1、ClGA20ox2、ClGA2ox1、ClGA2ox2和ClGA2ox3基因的正义表达载体，转化烟草，共获得转基因植株130株。从每个基因的阳性植株中随机挑选5个株系，经PCR、RT-PCR和Southern blotting鉴定出阳性株系3株。实时荧光定量PCR分析表明，在烟草对照植株中没有检测到目标基因表达，而在转基因植株中目标基因的表达量均显著提高，其中ClGA20ox1基因在转基因植株中最高表达量是149；ClGA20ox2基因的表达量达到144；ClGA2ox1基因的表达量达到217；ClGA2ox2基因的表达量达到71；ClGA2ox3基因的表达量达到258。同一基因在不同转基因株系中的表达量亦各不相同，随着ClGA20ox1和ClGA20ox2基因的表达量提高，转基因烟草植株节间伸长和株高增加等表型更加明显；随着ClGA2ox1和ClGA2ox3基因的表达量提高，转基因烟草植株矮化程度加剧。植株内源GA4含量测定结果显示转ClGA20ox1和ClGA20ox2基因的烟草植株叶片中GA4含量显著提高，而茎中GA4含量没有显著变化。转ClGA2ox1、ClGA2ox2和ClGA2ox3基因烟草植株叶片中GA4含量显著降低，其中转ClGA2ox3基因烟草植株降低最多。烟草对照植株经多效唑处理后，叶片中GA4含量显著下降，而茎中GA4含量无显著变化。这表明叶片中活性赤霉素的含量与植株的株型调控密切相关，ClGA20ox1和ClGA20ox2基因在转基因植株中过量表达，引起叶片中活性GA含量上升，从而促使植株伸长生长；ClGA2ox1和ClGA2ox3基因的过量表达，导致叶片中活性GA降低，从而诱导植株矮化；茎中的活性赤霉素水平对株型调控影响小。ClGA20ox1、ClGA20ox2基因促进植株叶肉细胞伸长，株高增加，节间伸长，始花期提前。转ClGA2ox1、ClGA2ox3基因抑制叶肉细胞伸长生长，叶片缩小，节间缩短，抑制花芽分化。另外，转ClGA2ox1、ClGA2ox2、ClGA2ox3和ClGA20ox2基因的烟草植株结实数量均少于对照烟草。转ClGA20ox1基因的烟草结实量与对照植株差异不显著。转ClGA2ox1基因烟草平均单果质量约为对照的一半，而转ClGA2ox3基因的烟草平均单果质量为对照的1.3倍；转ClGA2ox2、ClGA20ox1和ClGA20ox2基因的烟草植株平均单果质量与对照差异不显著。本研究从荔波连蕊茶叶片中分离出的ClGA2ox1基因不仅抑制植株伸长生长，而且抑制花瓣生长，能正常开花结实，这在花卉分子育种上具有重要的应用价值。ClGA2ox3基因不仅抑制植株伸长生长，而且平均单果质量显著提高，植株能正常开花结实，这对园艺植物分子育种具有重要的应用价值。ClGA20ox1和ClGA20ox2能显著促进植株增高，为将来利用RNA干扰或反义RNA等分子技术培育矮化植株奠定基础。更多还原

【Abstract】 GA20-oxidase and GA2-oxidase are two key enzymes in the metabolic pathway ofgibberellin synthesis, encoded by different multigene family, involved in the regulation ofplant endogenous GA activity level. GA20-oxidase and GA2-oxidase functions are mostlystudied in model plants. Their function in camellia plants have not been studied and whetherthey could regulate plant phenotypes has not been reported.In this study, two GA20-oxidase genes and three GA2-oxidase genes were cloned fromCamellia lipoensis Chang et Xu by means of homologous cloning method. They were namedClGA20ox1, ClGA20ox2, ClGA2ox1, ClGA2ox2and ClGA2ox3respectively. Bioinformaticsanalysis suggested that these sequences were GA20-oxidase or GA2-oxidase gene. The codingprotein of GA2-oxidase and GA20-oxidase were completely expressed in Escherichia ColiBL21induced by IPTG, indicating that the isolated genes had the ability to encode protein ineukaryotes.The results of quantitative Real time PCR analysis of GA20-oxidase and GA2-oxidasegene expression patterns were different significantly in different organs and growth periods ofC. lipoensis. Expression of ClGA20ox1and ClGA20ox2was low in the young stem, high instems and roots. Expression of ClGA20ox1in the seed was the highest, the lowest in the leaves.Expression of ClGA20ox2in the stem was the highest, the lowest in the young shoot tips. Itshowed that ClGA20ox1may be involved mainly in the regulation of plant growth anddevelopment of seed and root, ClGA20ox2may be involved mainly in the regulation of plantgrowth and development stem and leaves. ClGA2ox1, ClGA2ox2and ClGA2ox3were highlyexpressed in the mature leaves, the expression in the seed and leaves were low. ClGA2ox1expression in the stem was the highest, the lowest in the leaves. ClGA2ox2expression inmature leaves was the highest and the lowest in seed. ClGA2ox3expression in the root was thehighest and the lowest in the leaves. The results showed that ClGA2ox1may be involved in thegrowth and development of plant stem and leaves; ClGA2ox2may be involved in the matureleaf senescence metabolism; ClGA2ox3may be involved in the root growth. ClGA20ox1, ClGA20ox2, ClGA2ox1, ClGA2ox2and ClGA2ox3expression vectors wereconstructed into pCAMBIA1300expression vectors, transformed in Nicotiana tabacum. Atotal of130transgenic plants were obtained. Five transgenic plant lines were selectedrandomly from transgenic plants. Finally, three positive lines were obained by PCR, RT-PCRand Southern blotting. Quantitative Real-time PCR analysis showed that the target genes intransgenic expression were significantly increased compared with the control. The expressionamount of ClGA20ox1in transgenic plants was149higher than the control plant. ClGA20ox2expression amount was144higher than the control. ClGA2ox1expression amount was217higher than the control. ClGA2ox2expression amount was71higher than the control.ClGA2ox3expression amount was258higher than the control. Results showed that expressionof the same gene in different transgenic lines varied. With the expression of ClGA20ox1andClGA20ox2rose, the transgenic plants height increased obviously. The dwarf degree oftransgenic plants increased with the expression amount of ClGA2ox1and ClGA2ox3rose.Endogenous GA4content in the leaves of transgenic plants with ClGA20ox1orClGA20ox2was increased significantly while no significant changes in the stem. After treatingtransgenic plants with paclobutrazol, there was only a little change of GA4content in the stemand decreased significantly in the leaves. The GA4content reduced significantly in the leavesof transgenic plants with ClGA2ox1、ClGA2ox2or ClGA2ox3，specially decreased largely inthe transgenic plant with ClGA2ox3. After treating control plants with PAC, GA4content inthe leaves decreased while no significant change in the stem. It suggeted that phenotyperegulationg was closely related with the active GA level in the leaves. Thus, overexpression ofClGA2ox1or ClGA2ox3could induce dwarf phenotype and overexpression of ClGA20ox1orClGA20ox2could promote plant growth. The level of active GA in the stem has little effect onplant regulation.ClGA20ox1or ClGA20ox2can promote cells elongation, early flowering, plant heightand internode length increase. ClGA2ox1or ClGA2ox3could suppress cell elongation，reduceleaves and internodes. Flower bud differentiation was inhibited in the transgenic plant withClGA2ox1, ClGA2ox2or ClGA2ox3. In addition, fruit-bearing in transgenic plants withClGA20ox or ClGA2ox were lower than the control plant. Whereas there was no significantdifference in fruit-bearing between transgenic plant with ClGA20ox1had and the control plant. Meanwhile fruit mean weight of transgenic plant with ClGA2ox1was about half of the controlplant, while plants with ClGA2ox3was1.3times the height of the control plant. The fruitquality of transgenic plant with ClGA2ox2, ClGA20ox1or ClGA20ox2were similar to controlplant.In this study, ClGA2ox1not only inhibits stem elongation but also inhibits the growth offlowers petal. The transgenic plant with ClGA2ox1can normally flowering and fruit-bearing.ClGA2ox1has great value in molecular breeding. ClGA2ox3not only induces dwarf plant butalso increases the mean fruit weight, which has important application value in molecularbreeding. ClGA20ox1and ClGA20ox2can significantly promote the plant growth, which canbe used to cultivate dwarf plant by means of molecular technology, such as RNA interferenceand antisense RNA.更多还原