Cloning And Preliminary Functional Analysis Of Three Key Enzyme Genes Of Inulin Metabolism In Taraxacum Kok-saghyz Rodin

Taraxacum kok-saghyz Rodin（TK）is a perennial herbaceous plant of the genus Dandelion in Compositae.Its root contains high quality natural rubber（NR）.It is an ideal model plant for studying the biosynthesis of NR and a promising crop for NR production.However,low rubber content is the key factor limiting its commercial plantation.Inulin and NR are two important metabolites in the root.Both of them use sucrose as the substrate for their synthesis and compete with the same substrate.Taking the inulin metabolic pathway as a breakthrough may be an effective way to create TK lines with high rubber content.However the molecular mechanism of key genes of inulin metabolism in TK is still unclear.In this study,three key enzyme genes,Tk1-SST（encoding sucrose:sucrose 1-fructosyltransferase）,Tk1-FFT（encoding fructan:fructan1-fructosyltransferase）and Tk1-FEH（encoding fructan 1-exohydrolases）,were cloned in TK.The gene and protein characteristics were analyzed with bioinformatics softwares.The expression patterns of the three genes in different tissues and under different stress and hormone treatments were analyzed by qRT-PCR.The transgenic plants overexpressing Tk1-FEH gene were obtained by transgenic technique.CRISPR/Cas9gene editing technique was used to generate single gene knockout mutants（1-fft）and double knockout mutants（1-sst/1-fft）.The specific results are as follows:1 The key enzyme genes of inulin metabolism,i.e.Tk1-SST,Tk1-FFT and Tk1-FEH,were cloned,and their ORF contain 1908 bp,1869 bp and 1746 bp,encoding 635,622and 581 amino acids,respectively.Bioinformatics analysis showed that all of them belonged to the Glycosyl-hydrolase 32 family.Subcellular localization prediction analysis showed that all of them were located on Golgi apparatus.2 qRT-PCR analyzing revealed that the highest expression levels of Tk1-SST and Tk1-FFT were present in the roots,and the highest expression levels of Tk1-FEH were present in the leaves.All of them responded to PEG6000,mannitol stress,salt stress,Me JA,ET,and ABA treatments.The expression of Tk1-SST and Tk1-FFT were up-regulated,while the expression of Tk1-FEH was down-regulated under treatments.So the key enzyme genes of inulin metabolism are involved in the responses to abiotic stress and hormone signal transduction in TK.3 The plants overexpressing Tk1-FEH gene（including OE-FEH619-1,OE-FEH619-2 and OE-FEH20112-6）were generated by transgenic technique.The phenotype and drought tolerance of the overexpression lines were analyzed.Results showed that the biomass and drought tolerance of transgenic plants were significantly improved.The transcriptome of transgenic plants and their wild type after drought treatment was further analyzed.It was found that under drought stress,transgenic plants increase drought tolerance by ABA signal transduction,regulating ROS scavenging through Ca²⁺signaling pathways and related transcription factors,as well as upregulating genes expressions through electron transport chain of photosynthesis.4 CRISPR/Cas9 gene editing technology was used to knock out genes of key enzymes in inulin biosynthesis.TK1-FFT single knockout mutant（1-fft）and TK1-SST,TK1-FFT double knockout mutant（1-sst/1-fft）were generated using pYLCRISPR/Cas9Pubi-B vector system.Phenotypic analysis indicated that there was no significant difference between 1-fft plants with single gene knockout mutant and the wild type,but significant difference was present in the 1-sst/1-fft with double genes knockout mutant which petioles were shorter and the leaves were wider.In this study,the expression patterns of the key enzyme genes of inulin metabolism were analyzed.Tk1-FEH overexpression transgenic plants were generated,and found that overexpression of Tk1-FEH improve drought tolerance in TK.The single gene knockout mutant and double genes knockout mutant of inulin biosynthesis were obtained.The results of this study laid a good foundation for illuminating the mechanism of key enzyme genes of inulin metabolism in regulating inulin and natural rubber accumulation in TK.