Study On The Flower Bud Differentiation And Cloning Of Bud-formation Related MADS-box Genes In Cerasus Xueluoensis

Cerasus xueluoensis is a precious native resource with beautiful flowers.Unlike the triploid buds of Subg.Microcerasus and the solitary buds of Subg.Cerasus,Cerasus xueluoensis has the character of both triploid bud and solitary bud.It is of great significance to study the flower bud differentiation of Cerasus xueluoensis for understanding the flower bud differentiation of plants.However,the process and time of flower bud differentiation are still unclear which results in the molecular basis of symbiotic characteristics of Cerasus xueluoensis unknown.In order to solve this problems,the flower bud differentiation process of Cerasus xueluoensis is studied by improved paraffin section technology,and the specificity of Cerasus xueluoensis in flower bud morphological differentiation process is explored by comparing Cerasus tomentosa and Cerasus serrulata.Then two MADS-box genes which may lead to specific flower bud differentiation of Cerasus xueluoensis are cloned,and their temporal expression and organization in Cerasus xueluoensis are analyzed.The following results are obtained:1.The flower bud morphological differentiation process between Cerasus xueluoensis and that of Cerasus tomentosa and Cerasus serrulata is different.The flower bud differentiation process of Cerasus xueluoensis is divided into six stages:initial differentiation stage,inflorescence primordium differentiation stage,sepal differentiation stage,petal differentiation stage,stamen differentiation stage and pistil differentiation stage,which last nearly 102 days from early June to mid-September.By mid-September,pistil differentiation process is finished and enter dormancy stage.Compared with Cerasus serrulata,Cerasus xueluoensis has the same flower bud morphological differentiation process,but the differentiation time is shorter and the differentiation process starts earlier.Compared with Cerasus tomentosa,the flower bud differentiation time and duration of Cerasus xueluoensis are similar,but the differentiation stages is different.The differentiation process of Cerasus tomentosa doesn’t include the inflorescence primordium differentiation stage.2.The conserved domain and conserved motif analysis of CxlMADS1 and CxlMADS2indicates that the amino acid sequences encoded by M,I,K and C regions are typical MIKC MADS-box genes.The full length of CxlMADS1 and CxlMADS1 gene are 957 bp and 1274 bp,which contain open reading frame with length 636 bp and 708 bp.They encode 212 and 236amino acids,respectively.The theoretical molecular formula of amino acid sequences encoded by CxlMADS1 and CxlMADS2 are C₁₀₆₆H₁₇₅₅N₃₁₃O₃₃₈S₆ and C₁₁₂₁H₁₈₆₂N₃₂₀O₃₇₃S₁₃,the relative molecular masses are 24556.91 and 26207.82,the theoretical isopotential points are pI 6.86 and pI 5.17,the instability coefficients are 45.3 and 49.98,and the average hydrophilicity coefficients are-0.818 and-0.497,respectively.It indicates that CxlMADS1 and CxlMADS2gene belong to unstable alkaline and hydrophilic protein.The proteins encoded by CxlMADS1and CxlMADS2 genes contain a large number of alpha helix angles,irregular curls,beta folds and extension chains,accounting for 58.02%and 60.17%,28.3%and 11.02%,8.96%and4.72%,4.27%and 11.02%,respectively.3.The analysis of conserved domain and conserved motif shows that the C-terminal region controlling the function of gene protein of CxlMADS1 and CxlMADS2 is quite different,which indicates that the biological functions of the two genes are different.Homology analysis shows that CxlMADS1 and AGL6 have the closest genetic relationship,so they are classified as AGL6genes,which together with A,B,C and D genes regulates the development of four-round flower organs.CxlMADS2 has the highest similarity with the Dormancy-related gene DAM3 in Cerasus pseudocerasus.Therefore,CxlMADS2 is classified as DAM3 gene.DAM gene is a SVP homologous gene.It is reasonable to deduce that CxlMADS2 has similar function with SVP gene,determining the specificity of floral meristem and flowering time in early flowering stage.4.The analysis of gene expression of CxlMADS1 and CxlMADS2 in different tissues and organs shows that CxlMADS1 gene is only expressed in four-round flower organs,while CxlMADS2 gene is expressed in both leaves and four-round flower organs.The highest content of CxlMADS1 gene is found in leaves,and the lower content in petals and pistils.It can be seen that CxlMADS1 and CxlMADS2 genes are different in flower bud differentiation process and flower organ development stages.