Gene Cloning And Functional Validation Of Fructan Exonucleolytic Hydrolase (FEHs) In Helianthus Tuberosus L.

Jerusalem artichoke(Helianthus tuberosus L.)is a characteristic industrial crop on the plateau,which has attracted extensive attention in recent years.Fructan is the main storage material of Jerusalem artichoke,and it is also the main substances used for processing and utilization.The changes of fructan content and components are closely related to the processing quality during the whole storage period.A large number of studies have shown that FEH is the key enzyme gene for fructan degradation.Therefore,understanding the structure,function and expression characteristics of genes are important for adopting appropriate storage methods and improving storage resistance.In this manuscript,the homologous cloning and sequence analysis of the FEHs gene were carried out to understand the molecular structural of the FEHs gene of Jerusalem artichoke.The changes in fructan content and enzyme activity during storage stages and the later stage of blossom were measured and the correlation between the gene expression and the dynamics of fructan content changes was analysed in combination with the spatial and temporal expression characteristics of FEHs at different periods and the tissue expression characteristics at the same stage.Using VIGS technology to verify the degradation function of FEHs gene,and preliminarily clarify the function of Jerusalem artichoke FEHs gene at the molecular level.The main results are as follows.1.The genes of FEHI and FEHII were obtained by homologous cloning from "Qingyu No.1" which was a variety of Jerusalem artichoke,and the results revealed that the full length of FEHI and FEHII genes were 2,657 bp and 3,509 bp,respectively.The CDS were 1,683 bp and 1,746 bp,which presumably consisted of 5 introns and 6 exons.After bioinformatics analysis,the molecular weights of FEHI and FEHII proteins were predicted to be 62,785.04 Da and 64,811.48 Da,respectively,with the isoelectric point6.17 and 5.04.They have 3 conserved regions belonging to the 32 family of glycosyl hydrolases,which were hydrophilic proteins with extended structure and irregular coiling as the main secondary structures,and exercising function outside the plasma membrane.The genetic evolutionary relationships revealed that FEHⅠ and FEHⅡ were located in two major branches and were genetically distant.FEHI was the most genetically related to FEHI of chicory,FEHII was the most genetically related to cw INV of sunflower.2.The fructan content,enzyme activity and FEH expression levels in different tissues were measured during the late blooming period of Jerusalem artichoke.It was found that fructan content was highest in tubers,followed by stems,and tubers were mainly present with DP≥6 high polymerization fructan,while leaves accumulated the most DP<6 low polymerization fructans.FEH enzyme activity was detected in stems,leaves and tubers,with the highest enzyme activity in tubers.The expression of FEHⅠand FEHⅡ was the highest in leaves,and the expression of FEHⅡ was higher than that of FEHⅠ in leaves,tubers and flowers.Indicating that the expression of FEHⅠ and FEHⅡwas tissue-specific.The fructan content,enzyme activity and FEH expression levels in tubers were measured during the storage period of Jerusalem artichoke,and the results showed that the total fructan content of Jerusalem artichoke was exhibited a stable decreasing trend throughout the storage period.The fructan content of DP≥6 decreased rapidly after 14 d of storage,while the fructan content of DP<6 increased rapidly.The expressions of FEHⅠ and FEHⅡ showed the same trend during storage,both increased rapidly within 7 days of storage,after which the expression of FEHⅡ decreased slowly and the expression of FEHⅠ decreased rapidly.The enzymatic activity of FEHs appeared to increase at 91 days of storage.Indicating that at the beginning of storage of Jerusalem artichoke,FEHⅠ and FEHⅡ played a joint role in degradation,degrading high polymerization fructans into low polymerization fructans.The decrease of high degree of polymerization fructans and the increase of low degree of polymerization fructans make the change of total fructan content relatively stable.3.To verify the function of FEHs gene using virus-induced gene silencing(VIGS),the PDS-TRV2 vector was constructed by homologous recombination to verify the feasibility of VIGS technology system in Jerusalem artichoke,and injected into seedlings of Jerusalem artichoke.The leaves appeared yellow on the 7th day after injection,and chlorophyll content of the leaves was measured.The results showed that the chlorophyll content of etiolated leaves was significantly reduced relative to the control leaves,indicating that VIGS system was successfully constructed and played a role in Jerusalem artichoke.After that,the virus silencing vectors of Jerusalem artichoke fructan exohydrolase FEHI-TRV2 and FEHII-TRV2 were constructed and also injected into the seedlings of Jerusalem artichoke.When the leaves of PDS-TRV2-injected plants started to yellow,the silencing expression level of FEHs and the fructan content of the injected plants were measured.The results showed that the expression levels of FEHI and FEHII in the injected plants were lower than uninjected plants,and the fructan content was significantly higher than uninjected plants.It was speculated that the injection of bacterial solution silenced the FEHs gene causing their expression to be blocked and reduced,the fructans degradation function was weakened,and their fructan content was relatively high.At the molecular level,it was preliminarily determined that FEHI and FEHII genes of Jerusalem artichoke had fructan degradation function.