Cloning And Functional Analysis Of CpDRM3-1 And CpDRM3-2 Genes In Chimonanthus Praecox

As an auxin repressor superfamily,DRM1/ARP(Dormancy-Associated protein1/Auxin Repressed protein)gene family is closely related to the growth and development of plants,and plays an important role in biotic and abiotic responses of plants.It has been identified and studied in many monocot and dicot plants,but less in Chimonanthus praecox.In this study,using the c DNA obtained by RNA inversion of ’Concolor ’(Chimonanthus praecox ‘Concolor’),two DRM1/ARP family genes were cloned,which named CpDRM3-1 and CpDRM4-2.By analyzing the expression characteristics of CpDRM3-1 and CpDRM4-2 in different organs/tissues,different flower development stages,and the phenotypes of Arabidopsis thalana overexpressing CpDRM3-1 and CpDRM4-2 and their responses to drought and salt stress,we initially identified the functions of CPDRM3-1 and CPDRM4-2 genes in growth and development and abiotic stress.The main results are as follows:(1)By cloning and sequence analysis of CpDRM3-1 and CpDRM4-2 genes,it was found that CpDRM3-1 encoded 122 amino acids and its maximum ORF frame was 369 bp.CpDRM4-2 encoded 115 amino acids and had a maximum ORF of 348 bp.The promoter sequences of CpDRM3-1 and CpDRM4-2 genes contained many cisacting elements in response to drought,high salt and low temperature.In addition,the promoter sequences of CpDRM3-1 and CpDRM4-2 genes also contained hormone response elements,such as ABA,Me JA and so on.(2)The results of CpDRM3-1 and CpDRM4-2 expression analysis showed that the expression levels of CpDRM3-1 in roots were the highest,which were much higher than those in other tissues,about 130 times and 88 times higher than those in flowers and leaves,respectively.The gene was also expressed in reproductive organs,and had high expression levels in flowers,pistills and young fruits.It is speculated that this gene plays a positive role in regulating the reproductive growth of C.praecox,With the accumulation of low winter temperatures and the decay of plum blossom,the CpDRM3-1 gene is highly expressed.The expression level of CpDRM3-1 gene in flower buds of C.praecox and the expression level during dormancy-stage,late ovary maturation stage and low temperature accumulation stage was much higher than that in flower primordium formation stage and flowering stage,and the expression level of CPDRM3-1 gene in flower buds at the end of dormancy-stage and opening stage of Prunus chinensis was significantly decreased.The expression level of CpDRM4-2 was also extremely high in the root,which was42 times and 241 times higher than that in the flower and leaf,respectively.CpDRM4-2was also highly expressed in the flower,pistille,stamens,young fruit and other reproductive organs.CpDRM4-2 was induced by low temperature,and the expression level of CpDRM4-2 in flower buds increased gradually with the accumulation of low temperature.The expression level of CpDRM4-2 in flower buds at the stage of flower decay was the highest,suggesting that CpDRM4-2 gene might be involved in the regulation of reproductive growth.(3)Phenotypic analysis of CpDRM3-1 and CpDRM4-2 overexpressing Arabidopsis showed that35S::CpDRM3-1 and 35S::CpDRM4-2 overexpressing Arabidopsis promoted reproductive growth.Compared with the wild Arabidopsis WT,the bolting time and the first flower opening time were significantly earlier.The number and length of siliques increased.In terms of vegetative growth,the two genes over-expression were significantly smaller than the WT,the number of roseate leaves and leaf area were significantly reduced,the plant height was decreased,and the number of side branches was significantly lower than the WT lines,indicating that the two genes were involved in the regulation of vegetative growth of plants.Conclusion can be drawn from the above:CpDRM3-1 and CpDRM4-2 can promote reproductive growth and inhibit vegetative growth of overexpressed Arabidopsis.In addition,CpDRM3-1 and CpDRM4-2 could delay seed germination compared with the wild Arabidopsis WT,indicates that the two genes involved in seed dormancy;the expression levels of CpDRM3-1 and CpDRM4-2 were negatively correlated with hypocotyl elongation and root growth,suggesting that the functions of CpDRM3-1 and CpDRM4-2 might be related to plant abiotic stress.Overexpression of CpDRM3-1 and CpDRM4-2 reduced the drought and NaCl tolerance of transgenic Arabidopsis thaliana.These results above indicated that CpDRM3-1 and CpDRM4-2 genes could reduce plant height,reduce plant type and promote flowering of overexpressed Arabidopsis thaliana,and participate in the regulation of drought resistance and NaCl resistance.The functional study of CpDRM3-1 and CpDRM4-2 genes provides a research basis for the regulation of plant type and flowering stage,enriches the regulatory network of plant abiotic stress,provides some references for revealing the mechanism of winter flowering and dormancy-breaking of C.praecox,and also provides some guidance for the production and maintenance of C.praecox in garden applications.