Cloning And Function Identification Of Small GTP-binding Protein Gene CpRAC1 From Chimonanthus Preacox(Linn.) Link

Wintersweet, Chimonanthus praecox (Linn.) Link, is a famous traditional species originating from China, where it is appreciated as cut flower with high economic and ornamental value, owing to its important characteristics, such as blooming in deep winter, having strong fragrance and stress resistance et al. However, the regulation mechanisms of growth and development in wintersweet are not yet reported.Rac/Rop proteins belong to the subfamily of small GTPase, which play important roles as pivotal molecular switches in cell signal transduction. Going through the cycle between GTP-bound active form and GDP-bound inactive form, Rac/Rop proteins interact with various regulatory factors and downstream factors, respectively. They regulate different biological functions, including polar growth, disease and stress resistance, hormone signaling, production of reactive oxygen, and apoptosis.By analyzing the wintersweet ESTs, we found that the EST-DW0533 of the clone CH2097 was annotated as the small GTP-binding protein gene and its function in wintersweet was worth discussing. Therefore, the gene, named as CpRACl, was separated from the cDNA library of wintersweet flower. The expression vector was constructed and then transferred into Arabidopsis thaliana. The main results are as follows:1. The structure of CpRACl gene and features of its proteinBy random sequencing, the cDNA sequence of CpRACl was separated on the basis of EST analysis of constructed cDNA library. The cDNA sequence contains a 597bp open reading frame.The online prediction of ProtParam software indicated that the deduced CpRAC1 contains 198 amino acid residues, has a prediction molecular formula of C982H1563N261O283S7, with the molecular weight of 21.78kD, an isoelectric point (pI) of 9.32.The protein stability factor was 43.19, showing unstable protein character.Multiple sequence alignment by DNAstar 5.0 software revealed that CpRAC1 has high homology up to 94% with RAC proteins of Arabidopsis thaliana and Hevea brasiliensis, et al.. sharing some conserved structures, such as the GTP/GDP binding region, effector region, Rho insertion region, Serine/Threonine phosphorylation sites, basic amino acid region, C-terminal prenylation sites, et al.. Cluster analysis by MEGA4.1 showed that CpRACl belongs to class 1 RAC and is homologous with AtRAC3 of Arabidopsis thaliana.2. Expression analysis of CpRAClExpression features of CpRAC1 in various issues, florescences and under different treatments were analyzed by Quantitative Real-time PCR. The result proved that CpRACl had tissue specificity as the transcript was found in the root, stem, leaf and flower tissue, especially higher in the stamen. During different florescences, the expression of CpRAC1 was hardly detected in sprout period and display-petal period, but the highest in early senescence. In addition, it had a predominant expression in stamen during the early senescence and the senescence period. The treatment with H2O2 induced the expression of CpRACl. Exogenous ABA and gibberellic acid (GA3) treatment inhibited its expression. The results above indicated that CpRAC1 involved in signal transduction.3.Analysis of CpRAC1-transgenic Arabidopsis thalianaThe constructed expression vector pCAMBIA2301G-CpRAC1 was transferred into the agrobacterium EHA105, followed transforming Arabidopsis thaliana by inflorescence infection method. Sixteen T3 generation transgenic Arabidopsis thaliana were eventually gained after the process of Kan-resistance identification. Quantitative Real-time PCR was used to check the expression of CpRACl in the transgenic Arabidopsis thaliana. Finally, CpRAC1-16, CpRAC1-15 and CpRAC1-6 showing the highest, middle and lower expression of CpRAC1 gene, respectively, were selected to observe the phenotype and process some treatments.The observation of the transgenic and wild-type Arabidopsis thaliana showed that overxpression of CpRAC1 retarded the elongation, namely, postponing the senescence.Moreover, it was found that the seed setting rate declined because of the pollen germination delay and pollen tubes shorten, and the number of lateral branch arising from the stem increased.With exogenous ABA treatment, the transgenic Arabidopsis thaliana was more insensitive to ABA than the wild plants. Without GA3 treatment, the main root in transgenic plants lengthened and the number of lateral roots increased. The root growth in wild type plants with GA3 treatment almost reached the level of transgenic Arabidopsis thaliana without GA3 treatment. However, the transgenic plants were less insensitive to GA3 Over expression of CpRAC1 clearly improved the flavonoid content and chlorophyll content of leaves. The results illustrated that CpRACl may be involved in the regulation of ABA, GA3, flavonoids, et al., then affects the growth and development of plants.