Cloning,transformation And Functional Analysis Of DXS And FPS Gene In Tobacco

Terpenoids are widely distributed in various plants,and they demonstrate a broad structural diversity.Despite their important physiological and ecological roles in plant growth,development and defense,most of them are also of great pratical value.In recent years,the focus of research has been gradually shifted to the characterization of some key enzymes in the terpenoid biosynthetic pathway.These key enzymes are generally located at the critical branch points of the terpenoid biosynthetic pathway,and they catalyze the formation of some precursors and intermediates of the pathway,which subsequently determine the yield of subsequent products.Up regulation of genes encoding certain key enzymes can increase the yield of terpenoids.In this study,the key enzyme encoded by the genes DXS and FPS in the terpenoid biosynthetic pathway were cloned and analyzed using bioinformatics means.We also constructed three over-expressing constructs:p BI121-DXS?p BI121-FPS and p BI121-DXS-FPS,and those are used to transform tobacco plants.Through the analysis of those two genes expression and the solanesol content in the transgenic plants,explore the regulatory mechanism of solanesol metabolism in tobacco.The main findings are as follows:1.The complete coding region of 1-deoxy-xylulose-5-phosphate synthase(DXS)was cloned from the Nicotiana tabacum K326 for the first time,and its full length is2142 bp.The tobacco DXS gene,along with these from other plant species including wolfberry,potatoes,tomatoes,peppers and so on,was analyzed using bioinformatics tools,and the results shows that this gene is highly conserved with a sequence similarity score of 85% or higher when compared with other plant DXSs;This enzyme is a hydrophilic protein without any signal peptides or transmembrane domains,but it is predicted to be located in the chloroplast.It contains some conserved domains including TPP_synthase_N,TPP_PYR_DXS_TK_like,Transketolase_C and a multifunctional PLNO2582 sequence.Phylogenetic analysis indicates that the K326 DXS is grouped together with a variety of plant DXS2 family members in one big clade,and it is more closely related to the solanaceae plant DXS2 from an evolutionary perspective.Speculated that the gene sequence has the function of DXS2 genes and involves in the terpenoid metabolic process in tobacco.2.The complete coding region of farnesyl pyrophosphate synthase(FPS)was also cloned for the first time,and its length is 1029 bp.Bioinformatics analysis of this gene family was conducted,and the results are as follows: the sequence homology of the K326 FPS nucleic acid and protein sequences to tomato,peppers,South Africa,and so on is 85% or higher.The protein has a molecular weight of about 39.5 k D,and it is a stable hydrophilic protein without a signal peptide or a transmembrane structure.It has seven domains including a substrate binding site,a substrate-Mg2 + binding site,an active site,a chain length determining region,a catalytic site,etc.Phylogenetic analysis suggests that tobacco K326 FPS is closely associated with FPSs of pepper,tomato,potato and hypnotic sleeping eggplant,Solanaceae plants,which are clustered in a large clade with a branch confidence level of 100%,and the tobacco K326 FPS is clustered with FPSs of plant species from Solanaceae Nicotiana genus in a small clade,which is consistent with the phylogenetic relationship of plants.Presumably the gene should have the function of isopentenyl pyrophosphate synthase.3.The 3' regions of both the DXS gene and FPS gene were amplified and cloned using the RACE technique: the 3'RACE sequence of the DXS gene is 291 bp in length,while 3 'RACE amplification of the FPS gene resulted in two different sequences of235 bp and 261 bp.Results of the BLAST analysis suggest that both sequences are highly homologous to the sequences of other varieties in the Nicotiana tabacum species,and there is a gene sequence that is 100% identical to the sequencing sequence.Therefore,we believe that tobacco K326 possesses multiple FPS genes.4.Real-time PCR analysis of the tobacco DXS gene and the FPS gene shows that the two genes are expressed in roots,stems,leaves and flowers.The expression of these two genes in different tissues is ranked in this order: leaf> stem> root> flower.5.The two single gene over-expression constructs PBI121-DXS,PBI121-FPS and the double gene over-expression construct PBI121-DXS-FPS were successfully generated,genetic transformation of tobacco plants was carried out successfully.The number of transgenic tobacco plants obtained was 19 for plants over-expressing theDXS,23 for plants over-expressing FPS,and 8 for plants over-expressing the two genes.6.Expression analysis of the DXS gene and FPS gene in transgenic tobacco at fast growing stage shows that gene over-expressions have significant impacts on these two genes expression.Expression of these two genes in single gene transgenic plants was higher than that of the plants over-expressing double gene.At bud stage,the expression of DXS or FPS single genes in transgenic tobacco plants were significantly higher than those in transgenic plants.The co-expression of DXS and FPS genes in transgenic plants was consistent with that of single gene transformation.Expression of FPS gene in FPS transgenic plants or in double gene transgenic plants was higher than expression of DXS gene in DXS transgenic plants or in double gene transgenic plants,so FPS over-expression plants more accessible.At fast growing stage,the amount of solanesol was also significantly higher in single gene transgenic plants than that in non-transgenic tobacco,while there was no significant difference in terms of the solanesol content between the plants over-expressing both genes and non-transgenic tobacco.At bud stage,DXS or FPS single gene overexpression and double gene co-expression of transgenic plants increased the content of solanesol,but the double gene co-expression of transgenic plants was more significantly improved.So the metabolism of solanesol is affected by other factors such as growth period and gene interaction.At the same time,experiments show that accumulation of solanesol is not a simple positive correlation with gene expression in transgenic plants,and existing inconsistent case.The more complicated regulatory mechanism of solanesol metabolism will be further investigated.