Function Research Of GA20-oxidase And GA2-oxidase Genes From Camellia Lipoensis On Regulation Of Plant Phenotype

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.