Cloning And Functional Analysis Of PpCHS1 And PpSPL4 Genes Associated Flower Development In Kentucky Bluegrass

In the previous analysis of the A16 transcriptome of Kentucky Bluegrass dwarf mutants,we found that the levels of gene transcription on the pathways related to terpenoid biosynthesis,phytohormone metabolism,and secondary metabolites differed significantly from the wild type,in which flavonoids were synthesized.The 6 genes of the key genes in the CHS gene family all changed,with the most significant difference in PpCHSl,and the SPL gene family associated with flower development contained 5 genes,with PpSPL4 being the most significant difference.At the same time,this study found that the content of flavonoids in the mutants of Kentucky Bluegrass A16 was elevated,the flowering period was delayed,the seed color was deepened,and the saturation was increased.Therefore,two genes,PpCHS1 and PpSPL4,were selected as the research subjects.Based on the data of the transcriptome of Kentucky Bluegrass,the cDNA sequence of PpCHS1 gene was cloned.The gene contains a conserved region of chalcone synthase supergene family.The software analysis showed that the protein is a hydrophobic protein,type ? polyketide synthase,has a homodimer structure,subcellular localization results show that the protein encoded by the gene is located In cytoplasm.The expression analysis of PpCHS1 showed that this gene may participate in the synthesis of flavonoids in the flowering of Poa pratensis,and was induced by UV and high salt,and it was also continuously and highly expressed under the application of MeJA.The promoter region of the gene contains multiple core promoter elements TATA-box,CAAT-box,light regulatory related elements,phytohormone regulatory elements,and the MYB transcription factor transactivator-related regulation associated with drought induction and flavonoid synthesis.Sequences,etc.This indicates that the gene is responsive to light,phytohormones,and MYB transcription factors.The PpCHSl gene plant expression vector was constructed and transformed into Arabidopsis thaliana.The growth and development status of the obtained T2 transgenic plants showed no abnormal changes,but the content of flavonoids in the body increased.This indicates that the cloned PpCHS1 gene has the biochemical function of chalcone synthase,but the specific biological function needs further study.The test cloned PpSPL4 gene.The gene contains a conserved region of the SBP gene family,and the similarity to other plant SBP genes is above 67.83%.Its open reading frame is 961 bp and encodes 326 amino acids with an isoelectric point of 9.07.High-level structural analysis shows that the DNA-binding protein has the highest similarity to AtsSPL4 gene.Subcellular localization results show that the protein encoded by this gene is located in the nucleus,and the grass is premature.The analysis of PpSPL4 gene expression in rhizomes,roots,stems,leaves and different flowering stages showed that there was a significant difference in the relative expression of PpSPL4 genes in different tissues of Kentucky Bluegrass.The relative expression level was highest in flowers,and the PpSPL4 gene was affected by GA and sucrose.The induction indicated that the gene may be a flower bud differentiation and transcription factor related to the GA pathway during flower development of Kentucky Bluegrass.The SPL gene family has the function of regulating the synthesis of flavonoids,but whether PpSPL4 gene has a response function in Poa pratensis,needs further study.Expression analysis showed that both PpCHSl and PpSPL4 were involved in the flower development of P.virens.Studies of PpCHSl and PpSPL4 genes could provide a theoretical basis for the study of seed breeding mechanism and breeding of Kentucky Bluegrass.