Cloning And Functional Analysis Of PbMADS12 And PbMADS14 Genes In Pear(Pyrus Bretschneideri Rehd.cv. 'Dangshansuli')

Pyrus bretschneideri Rehd.cv.‘Dangshansuli' fruits which lied between Pyrus bretschneideri and Pyrus pyrifolia.in horticultural classification,and it has a long cultivation history in DangShan county.It's one of the largest varieties of cultivated area in China.In recent years,there was a naturally mutation with high-content sugar in pear fruits which were found from germplasm resources of P.bretschneideri ‘Dangshansuli'.The total sugar content of the mutant fruit was significantly increased.The fructose which have a high sweetness was the main component of sugar.The fruit surface was roughness,had many fruit rust and dense lenticels.The wild-type fruit is emerald green.The variation characteristics are very stable.We further did a comparative study of metabolomics and transcriptomics of the mutant and wild type fruits,and found that significant changes in the metabolism of ethylene during mutant fruit development.We hypothesized that the regulation of upstream two MADS transcription factors related to this.For this reason,this paper of cloning two MADS transcription factor gene and did functional analysis,to verify the regulation of MADS to Ethylene Metabolism in ‘Dangshansuli' pear fruit development.We provided the experimental basis for the interpretation of the molecular mechanisms of high glucose ‘Dangshansuli' pear fruit sugar accumulation in mutant.At the same time,studies on fruit ripening has more concentrated in the climacteric fruit,less for non-climacteric fruit.Regulation on ‘Dangshansuli' pear fruit ripening process related genes has important research significance.In this experiment,‘Dangshansuli' pear fruit in wild-type and mutant varieties were used as test material.According to the measured data in transcriptome and metabolome,we cloned the two MADS genes named PbMADS12 and PbMADS14,and did some bioinformatics analysis selected thirteen ethylene biosynthesis-related unigenes.We using RT-PCR to analyze the expression pattern of thirteen unigenes.We had construced two overexpression vector which named PC1301-MADS12 and PC1301-MADS14.And we transformed them into Micro-Tom tomato cotyledon to verify its functionality.The experimental study achieved the following main results:1.The variation and non-variation fruits of third periods(DPA100)were used as test materials.Two genes named PbMADS12 and PbMADS14 were cloned.The sequencing results showed that there was substantially no difference in two MADS genes between the variation and non-variation fruits.The ORF of PbMADS12 composed of 720 bp,and encoding 239 amino acids.The ORF of PbMADS12 composed of 750 bp,and encoding 249 amino acids.It showed that the mutation is not caused by the two genes.2.The MADS gene sequences and the phylogenetic tree analysis showed that the two MADS genes and the genes from NCBI have high homology.Both of them contain two conserved domains named MADS-box and K-box.3.The genes contained PbMADS12 and PbMADS14 in ethylene biosynthesis from‘Dangshansuli' pear transcriptome analysis were did real-time quantitative PCR analysis.The result showed that the peak value of gene expression in the non-variation fruits was in DPA 130,the peak value of gene expression in the variation fruits was in DPA 160.4.Over expression vector named PC1301-MADS12 and PC1301-MADS14 were successfully constructed and transformed to Micro-Tom tomato.Identification by PCR with the primers of PbMADS12,PbMADS14 and Hyg gene.Five regenerated plants were obtained.