| Yam(Dioscorea opposita Thunb.)is a cash crop with great market prospects and industrial development potential.The research on its yield and quality mechanism is receiving increasing attention from scholars at home and abroad.Starch is the main storage material of yam tubers,accounting for more than 65% of the dry matter content,which directly affects the yield and quality of yam.Starch-sugar interconversion affects the growth and developmental processes of yam.Therefore,the analysis of starch-sucrose metabolism involved in the regulation of signaling and substance synthesis during yam tuber formation and expansion can provide an important theoretical basis for yam tuber yield formation and quality genetic improvement.In this study,Bikeqi(B1)and Dahechangyu(DHCY)were used as test materials.Morphological indicators and tuber contents of amylose,amylopectin,starch,soluble sugars,reducing sugars,and sucrose,as well as key enzyme(BAM,ISA,SS,AMY,SPS,SUS)activities of starch-sucrose metabolism of the tubers of the two yam varieties were measured and analyzed,respectively.Untargeted metabolomics was used to detect and analyze all metabolites within the tubers of the two yam varieties at three expansion periods.We further combined analysis of transcriptomics and metabonomics to screen key differentially expressed genes and metabolic pathways.The full-length c DNAs of DoBAM1 and DoSS2,key genes of starch-sucrose metabolism,were cloned by RACE technique,and the ORFs of DoISA3,a key gene of starch-sucrose metabolism,were cloned by RT-PCR technique,and the ORFs of the three genes were analyzed bioinformatically.The transient expression vectors of DoBAM1,DoISA3,and DoSS2 were constructed and injected into tobacco leaves for subcellular localization.We used q RT-PCR technology to analyze gene expression in different growth and development stages and tissues of yam tubers,and analyzed the correlation between gene expression levels and starch,sugar content,and key enzyme activities in the tubers.The plant expression vector of the DoBAM1 gene was constructed and transformed on tobacco leaves to identify its function.The main findings are as follows:1.B1 and DHCY yam tuber starch and its fraction content showed a trend of increasing during T1-T3 period and then decreasing,while sugar content was highly significantly negatively correlated with starch content.BAM and AMY may play important roles in regulating starch hydrolysis and sugar synthesis.The correlation coefficient between SS and amylopectin was the highest,and the accumulation of starch and amylopectin may be regulated by it.SPS and SUS were positively correlated with sugar content,indicating a possible positive regulation of sugar accumulation.2.The diversity and variation of metabolites among the two yam varieties and at different expansion stages were analyzed using untargeted metabolomics.550 metabolites(pos mode)and 624 metabolites(neg mode)were identified,and the number of differential metabolites enriched in each period and each variety was different,and the highest number of enriched metabolites were D＿150 and B＿150,and the number of differential metabolites was higher in DHCY than B1.Based on cluster analysis,amino acids and their derivatives,organic acids,and sugars were found to be up-regulated during tuber expansion.The main enriched metabolic pathways were lipid metabolism,amino acid metabolism,carbohydrate metabolism,and secondary metabolite biosynthesis pathways.3.The combined transcriptomic and metabolomic analyses revealed that the main enriched metabolic pathways of the two yam varieties were 14 carbohydrate metabolism,13 amino acid metabolism,and 10 lipid metabolism,among which the more significant enriched pathways in carbohydrate metabolism were starch and sucrose metabolism,glycolysis/gluconeogenesis and pyruvate metabolism.The expression of relevant differential genes involved in starch-sucrose metabolism plays a positive or negative role in regulating differential metabolites.Among them,UGD5,BAM1,SUS2,TPP6,SS2,ISA3,CIN3,FRK1,SBE1,HXK1,and APL2 genes play key regulatory roles in the starch-sucrose metabolic pathway.4.Based on the combined transcriptome and metabolome analysis,the key genes of starch-sucrose metabolism DoBAM1,DoSS2,and DoISA3 were screened,and the full-length c DNAs of DoBAM1 and DoSS2 were cloned.The full-length c DNA of DoBAM1 was 2806 bp and the ORF length was 1629 bp.The full-length c DNA of DoSS2 is2939 bp and the ORF length is 2325 bp.The ORF of DoISA3 was cloned and is 1584 bp.Bioinformatics analysis revealed a highly conserved Glyco＿hydro＿14 structural domain and catalytic residues Glu-186 and Glu-380 in DoBAM1.DoBAM1 showed the highest homology with Da BAM5 of Dioscorea alata.DoISA3 contains the Amy Ac＿Glg＿debranch structural domain.DoISA3 clusters in the same branch with the ISA3 proteins of Dioscorea alata and Dioscorea cayenensis subsp.Rotundata,which are also in the Dioscoreaceae family.DoSS2 is most closely related to Da SS2 of Dioscorea alata and As SS2 of Asparagus officinalis L.Subcellular localization results indicated that both DoBAM1 and DoISA3 were localized to the cytoplasm and cell membrane,DoSS2 is localized in chloroplasts.5.The expression patterns of DoBAM1,DoISA3,and DoSS2 genes were analyzed.3genes were specifically expressed in different tissues of yam,and DoBAM1 was more expressed in leaves of 2 varieties than in stems and tubers;DoISA3 was expressed in tubers > leaves > stems in B1,and in stems > leaves > tubers in DHCY;DoSS2 was more expressed in leaves of 2 varieties than in stems and tubers.The expression of DoBAM1 was positively correlated with starch and sugar content and highly significantly and positively correlated with BAM activity.DoISA3 gene expression was negatively correlated with starch content and ISA activity,and positively correlated with sugar content.DoSS2 gene expression was highly significantly and positively correlated with amylopectin,starch content,and SS activity,and the correlation coefficient with amylopectin was higher than that with starch content and amylose content.6.The DoBAM1 gene was transferred into tobacco for functional analysis,and the expression of DoBAM1,chlorophyll,soluble sugar,and BAM activity in the transgenic strain were significantly higher than WT,and the starch content was significantly lower than WT when the transgenic tobacco and wild-type tobacco was treated with different levels of cold stress.The amount of reactive oxygen species accumulated in the transgenic strain was less.The SOD,POD,and CAT activities were significantly higher than WT,and the MDA content was significantly lower than in the WT plants.These results demonstrate that the DoBAM1-transformed plants could improve cold tolerance by regulating soluble sugar levels and antioxidant capacity. |
