Cloning And Functional Study Of Marigold GA2ox Gene

Marigolds are widely used in gardens and landscape.Compared with tall varieties,dwarf and compact marigold are preferred by gardeners.In this study,marigold strain ‘c-16’ was used as material.Gene GA2 ox was cloned by homologous cloning.Full length of GA2 ox is 1247 bp,and it encodes 328 amino acids.It is closely related with Mikania micrantha Kunth and other composite plants.Bioinformatic analysis showed that the total number of atoms of marigold GA2 ox gene was 5193 and the isoelectric point was 6.62.The encoded protein was presumed to be a stable protein with partial hydrophilicity.No signal peptide was detected.Meanwhile,it doesn’t havetransmembrane structure and obvious hydrophobicityregion.Subcellular localization successfully localized the gene in the cell nucleus and cytoplasm.The results of spatial and temporal expression pattern of GA2 ox showed that it expressed in different tissues but the expression levelwas significantly different.The expression level of GA2 ox was highest in the stem,followed by leaf and root.Its expression level was lowest in mature dormant seeds.It indicated that GA2 ox mainly regulate the growth and development of stem and leaf.It has little regulatory effect during the seed dormancy process of marigold.Analysis of expression level of GA2 ox in different growth period showed that the expression level of this gene increased with the growth of the plant.It increased rapidly between the 6-leaves to 8-leaves stage and reached the maximum expression,it was much higher than other growth stages.After that,its expression began to decline.At the 8-leaves stage,marigold plants developed from vegetative growth to reproductive growth.The expression of GA2 ox was highest at the 8-leaves stage,which inactivated GA and inhibited the growth of marigold.The cloned GA2 ox gene encoded protein which can be completely expressed in E.coli and has the ability to encode proteins in prokaryotes.The positive-sense expression vector of GA2 ox was constructed using binary vector PRI-101-AN.The expression vector was introduced into Agrobacterium GV3101 and transformed into tobacco.Four positive strains were identified by antibiotic screening and RT-PCR.In this study,gene GA2 ox was successfully isolated from marigold,and its bioinformatics analysis and subcellular localization were carried out.Through the expression analysis of GA2 ox and the analysis of transformed plants,the function of GA2 ox on plant growth and development was further explored.This study provided research materials and scientific support for character improvement of marigold,and played an important role in promoting the process of marigold dwarf breeding.It also laid the foundation for future application of molecular techniques such as RNA interference or antisense RNA inbreeding of dwarf plants.