Cloning And Expression Analysis Of MADS-box Gene Related To Floral Organ Development In Paeonia Suffruticosa

Paeonia suffruticosa is one of the most famous traditional flowers in China,which has high ornamental commercial and medicinal value.In the long process of cultivation and evolution,the peony’s flower structure and color have complex changes.In recent years,by using plant molecular breeding technology to cultivate new peony varieties has become one of the most hot-pot research for many scholars.The MADS-box gene family is the key to regulate the floral organ morphogenesis of plants,but the research on the MADS-box gene of peony is relatively insufficiency at present.Therefore,based on the database of peony transcriptome from our research group,six MADS box genes that may be involved in the development of peony flowers were screened.The full length of six genes was cloned by homologous cloning method,and these genes were also analyzed by bioinformatics method.Using q RT-PCR and In-situ hybridization to analyze and identify the expression patterns of these six genes in different stages of flower bud differentiation.The main results are listed as following:(1)Six full-length c DNA of flower development related genes were cloned from petals of Peony ’Luo Yanghong’,named as: Ps AP150(Gen Bank Accession No.:MT225536);Ps AP185(Gen Bank Accession No.:MT225538);Ps PI72(Gen Bank Accession No.:MT225533);Ps AP353(Gen Bank Accession No.:MT225537);Ps AG69(Gen Bank Accession No.:MT225534);Ps SEP1(Gen Bank Accession No.:MT225535).The sequence length are 768 bp;729 bp;636 bp;609 bp;729 bp;729 bp and the encoding amino acids number are 255;242;212;203;243;243.The results of sequence comparison showed that all the six genes contained MADS domain,K domain and corresponding characteristic region,belonging to MADS-box gene family in peony.The secondary structure of six genes are contained α-helix,β-fold,extended chain and random curl,all of which are characterized as unstable hydrophilic proteins.(2)The result of six genes expression in different bud differentiation stages from peony showed that the expression patterns of six genes in two different flower types of peony were similar but slightly different,and they were expressed in all stages of peony flower development.The expression of Ps AP150 in sepal differentiation stage of ‘Xue Ta’ and ‘Feng Danbai’ was significantly increased,which was 32.41 times and 27.82 times than the expression at the pre-stage of bud differentiation,respectively.The expression of Ps AP185 in the petal and stamen differentiation stage of ‘Xue Ta’ was much higher than other four stages,and reached the highest level in the petal differentiation stage of ‘Feng Danbai’.The expression of Ps PI72 gene was 22.37 times higher than the pre-stage of bud differentiation.The expression of Ps AP353 gene was the highest at the stamen differentiation stage from ‘Xue Ta’ and ‘Feng Danbai’.The expression of Ps AG69 gene has increased in six stages of flower development,and reached the highest level in pistil differentiation.The expression ofPs SEP1 gene reached the maximum at the sepal differentiation stage from ‘Xue Ta’ and ‘Feng Danbai’.(3)The results of In-situ hybridization from ‘Feng Danbai’ showed that Ps AP150 gene and Ps SEP1 gene were located in bract,and Ps AP185 gene was located in sepal.And Ps PI72 gene and Ps AP353 gene were located in petal,and there was no hybridization signal of Ps AG69 gene in the whole process of flower development.In ‘Xue Ta’,Ps AP150 gene and Ps SEP1 gene were located in sepal,Ps AP185 gene and Ps PI72 gene were located in petal,and Ps AG353 gene might be located in stamens.In conclusion,the six genes cloned in this study were all belonged to the MADS-box gene family,and their secondary structure and properties are similar.Ps AP150 gene may be involved in the regulation of calyx differentiation of peony,Ps AP185 and Ps PI72 gene may be involved in petal differentiation,Ps AG353 gene may be involved in stamen differentiation,and Ps SEP1 gene may be involved in calyx differentiation.The results provide a theoretical basis for using molecular biology to understand the role of MADS-box gene in the development of peony floral organs,and lay a theoretical foundation for molecular breeding of peony flower type and directional improvement of new peony varieties.