Analysis Of Enhancer Sequence Based On Differential Expression Of VInv Gene And Relationship Between The Enhancer And Sugar Metabolism In Potato

Sugar metabolism is the central metabolism in the body of whole plant,it can be linked to multiple metabolic pathways such as nucleic acid metabolism,lipid metabolism,protein metabolism and secondary metabolism,and its complex metabolic processes run through the whole process of plant growth and development.Sugar metabolism related enzymes and their coding genes play important roles in potato starch-sucrose metabolic pathway.In which vacuolar invertase(VINV)is not only related to sugar metabolism and transport,but also has relationship with plant growth and development.The VINV enzyme produces a necessary sucrose concentration gradient to facilitate loading and unloading of the phloem.The VINV enzyme hydrolyzes sucrose to glucose and fructose,which greatly increase osmotic pressure of cells,and thereby supply driving force for cell elongation.In our previous study,the second intron of vacuolar invertase gene(Vlnv)was confirmed to have function of cold-induced enhancer in potato.In this study,we further analyzed the effect of sequence variation of the enhancer on Vlnv gene expression,and the correlation for gene expression among those genes associated with sugar metabolism related enzymes after cold stress,so as to reveal the relationship between the cold-induced enhancer and potato sugar metabolism,which will lay a foundation for molecular mechanism of tolerance to cold stress in potato.The main results were as follows:I.A PCR-based homologous cloning strategy was used to clone the second intron of the Vlnv gene in 38 potato germplasm resource materials with different ploidies(diploid,tetraploid,hexaploid),and the number and distribution of allelic variants in each material were analyzed.The results showed that 2 of 31 diploid materials contained only one type of allelic variants,and the other 29 diploid materials had two types of allelic variants(a,b);All three tetraploid materials contained three types of allelic variants(a,b,c);1 of 4 hexaploid materials contained three types of allelic variants(a,b,c),and the others had four types of allelic variants(a,b,c,d).II.Bioinformatics analysis was subsequently performed on intron allele sequences of the Vlnv gene cloned from the 38 potato germplasm resource materials and 2 diploid control clones(RC-7 and RH).The results of multiple sequence alignment showed that the similarity of the intron sequences between potato germplasm resource material C9 and the negative control clone RC-7 was higher than those between C9 and other 37 potato germplasm resource materials which had higher similarity with the positive control clone RH.After further combined with analysis of phylogenetic tree,the results revealed that genetic relationship of the intron between C9 and RC-7 was closer than those between C9 and the others,while the genetic relationship of the intron between the other 37 germplasm resource materials and RH was closer than those between the other 37 germplasm resource materials and RC-7.?.Nine wild diploid materials randomly selected from 31 diploid potato germplasm resource materials and 2 diploid potato control clones(RC-7 and RH)were used for further study on expression of sugar metabolism related enzyme genes.It was found that genes VInv and AGPase,which involved in sucrose hydrolysis and starch synthesis,respectively,were normally expressed in the stem tissues,and their overall expression levels were much higher than those of genes SPS and SuSy related to sucrose synthesis or decomposition,and the genes SPS and SuSy almost silenced in the stem after cold stress.Positive linear correlation(r=0.623,p<0.001)for relative gene expression levels was only found between genes Vlnv and AGPase,which indicated that these two genes were co-expressed in the stem after cold stress.Analysis of the multiple sequence alignment and the phylogenetic tree were subsequently conducted on enhancer alleles from the 11 diploid materials.Nine differential DNA sites were detected between the negative control clone RC-7 and the others(included the positive control clone RH),and the genetic relationship of the enhancer between clone RC-7 and the others was far away.Based on the above results together with the expression models of the VInv gene in the 11 diploid materials,we concluded that the sequence variation of the enhancer might affect the VInv gene expression,and that the enhancer might play an important role in potato sugar metabolism after cold stress.