| Watermelon,as a popular summer fruit,is very popular and is an essential tool for relieving heat in every household.In the market,the flavor of watermelon determines its price and sales,and the pulp hardness is an important factor affecting the taste of waterme lon.Therefore,in order to pursue a higher quality taste,it is imperative to study the pulp hardness of watermelon.The MADS-box gene family widely exists in monocotyledonous and dicotyledonous plants,which is an indispensable member of growth and development.Initially,it was found that many members of the MADS-box family were involved in the development of flower organs,and on this basis,an ABCDE flower development model was established.In recent years,more and more articles have reported that the MADS-box family genes are involved in the regulation of fruit development and ripening,with the most relevant articles using the model crop tomato.In this paper,wild watermelon(Citrullus lanatus var.lanatus)“51” and cultivated watermelon(Citrullus lanatus cultivar)“ 1338” were used as experimental plant materials.Based on the watermelon fruit development transcriptome and real-time fluorescence quantitative PCR,the gene ClMADS37 related to the firmness formation of watermelon pulp was identified.Th e gene was cloned in the above watermelon,and the gene characteristics were analyzed,spatio-temporal expression characteristics and subcellular localization were analyzed,and the overexpression vector was constructed.To explore the relationship between ClMADS37 gene and watermelon pulp hardness formation in tomato variety AC(Solanum lycopersicum Mill.var.Ailsa Craig),which has certain significance for the utilization of MADS-box gene and the improvement of watermelon germplasm resources.The main findings of this study are as follows:(1)By analyzing the transcriptome data of firm and soft watermelon(Clfh),we found that the relative expression of ClMADS37 gene in firm watermelon(Clfh)was significantly higher than that in soft watermelon(Clfh)at the same time after pollination,and the hardness of the fruit decreased with the increase of pollination days.The relative expression of ClMADS37 gene was also reduced.It is speculated that this gene positively regulates watermelon pulp hardness and participates in the regulatory pathway of watermelon pulp hardness formation.(2)To test this hypothesis,the spatiotemporal expression characteristics of ClMADS37 gene were analyzed by q RT-PCR,and the results showed that the expression of ClMADS37 gene decreased with the increase of pollination days.During the same period,the expression of this gene in hard flesh watermelon “51” was significantly higher than that in soft flesh watermelon“1338”.The results showed that the ClMADS37 gene was positively correlated with pulp stiffness.The relative expression of ClMADS37 gene in watermelon material “51” and “1338” was the highest in the stem,and the lowest in the leaf.The relative expression of ClMADS37 gene in all parts of watermelon material “1338” was higher than that of watermelon material “51”.(3)The central pulp c DNA of watermelon material “51” and “1338” was used as template,and specific primers were designed for PCR amplification.The full-length CDS of ClMADS37 gene was 1176 bp,encoding 391 amino acids,and its molecular weight was 43092.83 k D.The theoretical isoelectric point was 4.63,which was an acidic protein.The total number of negatively charged residues and positively charged residues were 63 and 36,respectively,and the instability index was 33.85,which belonged to the stable protein.The aliphatic index was 74.53.The coefficient of hydrophilicity is 0.653,which belongs to hydrophilic protein.In the secondary structure of ClMADS37 protein,the α helix accounted for 42.2%,the β turn accounted for6.14%,the extended strand accounted for 9.21%,and the random coil accounted for 42.46%.Phylogenetic tree analysis showed that the protein encoded by ClMADS37 had the highest homology with melon and the closest evolutionary distance.The results of cis-element analysis of the promoter showed that the transcription and protein expression of the gene were intrinsically re lated to light response,hormone induction and respiration.The subcellular vector of ClMADS37 protein was instantaneously expressed in tobacco leaves.After the transformation of the recombinant vector of p35+ ClMADS37-GFP,clear green fluorescence was observed in the nucleus of the tobacco fusion protein,in comparison with the control group(P35-GFP),confirming that the ClMADS37 gene was located in the nucleus.(4)The overexpression vector of ClMADS37 was constructed and transformed into tomato cotyledon by agrobacterium tumeticum staining method to obtain tomato transformed seedlings.After PCR verification,7 positive seedlings were obtained,and the q RT-PCR test of ClMADS37 gene in positive seedlings showed that 4 of them had extremely significant up-regulation of ClMADS37 gene expression. |
