| The rolgene has been widely studied as a target gene for improving the secondary metabolism of plants.Among them,the rolB and rolC genes are powerful target genes in the rolgene family,and the promotion effect is different in different plants.Therefore,different plants have different the main effect of rolgene,and there is a certain mutual influence between different rolgenes.In this study,A.belladonna was used as experimental material to construct a plant expression vector that overexpressed rolB gene and co-expressed rolB and rolC genes,and obtained transgenic A.belladonna through genetic transformation,and then combined with the p BI121-rolC A.belladonna overexpressing rolC gene obtained in the laboratory earlier.To explore the effect of rolB and rolC genes on the TAs content of A.belladonna by analyzing the horticultural shape,tropane alkaloids(TAs)content,TAs synthesis pathway gene expression,and H2O2 and NO content of three transgenic A.belladonna and the interaction between rolB and rolC genes,laying a foundation for exploring the mechanism of rolgene regulating the synthesis of secondary metabolites,and also providing a new method for further research to promote the anabolic engineering of A.belladonna tropane alkaloids.The results of the study are as follows:(1)Establishment and molecular detection of plants overexpressing rolB and rolC genes: In this study,a plant expression vector p BI121-rolB overexpressing rolB gene and a plant expression vector p1305NKB+121C coexpressing rolB and rolC genes were constructed.Infected A.belladonna cotyledons and hypocotyls to obtain p BI121-rolB A.belladonna overexpressing rolB gene and p1305NKB+121C A.belladonna co-expressing rolB and rolC genes,combined with p BI121-rolC gene overexpression obtained in the laboratory rolC A.belladonna as follow-up research material.(2)Effects of rolgene on horticultural traits of A.belladonna: pBI121-rolB A.belladonna showed dark green leaves,increased leaf area,increased flower shape and tended to altar-shaped corolla,decreased pistil height,fertile pollen but decreased vigor,root system Developed,dense and other phenotypes;p BI121-rolC A.belladonna showed decreased apical dominance,earlier flowering time,increased number of flowers,smaller flower shape,sterile pollen,lush axillary buds,shortened internodes,light green leaves with long and narrow leaves,root system The phenotype of P1305NKB+121C A.belladonna is more inclined to p BI121-rolC A.belladonna,but the flower shape,leaf and plant height are smaller,the number of branches and leaves are more,and the root system is senescent earlier,and the rest of the phenotypes are similar to p BI121-rolC.Eggplant consistent.The results of horticultural trait analysis showed that exogenous introduction of rolB and rolC genes could cause developmental variation in the aerial and underground parts of A.belladonna plants.(3)The effect of rolgene on the content of TAs in A.belladonna: the contents of hyoscyamine,anisodamine and scopolamine in the stems of A.belladonna in p BI121-rolB were increased by 1.44-1.86 times,1.49-2.87 times and 4.49-4.77 times,respectively.There was no significant difference in alkaloid content compared with wild-type A.belladonna.The contents of hyoscyamine,anisodamine and scopolamine in p BI121-rolB A.belladonna leaves were increased by 1.54-2.70 times,1.90-2.93 times and 1.44-1.73 times,respectively,while only the C-1 line in the stem had significantly higher TAs content than Compared with the control,its content was increased by 3.65,4.66 and 6.35 times,respectively.However,the content of TAs in the leaves of p1305NKB+121C A.belladonna was not significantly different from that of the wild-type A.belladonna.Except for the BC-2 line,the other lines in the stem had no significant difference with the wild-type.The contents of scopolamine,anisodamine and scopolamine in the BC-2 strain were increased by 2.5 times,3.39 times and 5.04 times,respectively.The analysis results showed that both rolB and rolC genes increased the TAs content in A.belladonna.rolB gene mainly promoted the accumulation of TAs in A.belladonna stems,while rolC gene mainly promoted the accumulation of TAs in A.belladonna leaves,and the overall promotion effect of rolC gene was greater than that of rolB gene.The improvement is not significant.Therefore,the rolC gene was the main gene of A.belladonna,and the introduction of the rolB gene weakened the promoting effect of the rolC gene on the TAs content of A.belladonna to a certain extent.(4)TAs synthesis pathway gene expression analysis: In the roots of p BI121-rolB A.belladonna and p BI121-rolC A.belladonna,the expression levels of TAs synthesis pathway genes UGT I and H6 H were significantly up-regulated,and the expression level of TR II was significantly down-regulated.In p BI121-rolB A.belladonna,the expressions of UGT I and H6 H were up-regulated by 14.30 and 11.80 times,respectively,while the expression of TR II was down-regulated by 5.71 times.In p BI121-rolC A.belladonna,UGT I and H6 H were up-regulated by 55.63 and 11.53 times,respectively,while the expression of TR II was down-regulated by 7.35 times.The expression levels of UGT Ⅰ,H6 H and TR Ⅱ were inconsistent among the A.belladonna strains of p1305NKB+121C.The analysis results showed that the rolB and rolC genes increased the TAs content of A.belladonna mainly by up-regulating the expression levels of UGT I and H6 H and down-regulating the expression levels of TR II.(5)Analysis of H2O2 and NO content in A.belladonna: In this study,in order to explore the mechanism of rolgene regulating the accumulation of TAs,the H2O2 and NO content in three transgenic A.belladonna were detected.It was found that the introduction of rolB gene and rolC gene did not cause significant changes in H2O2 and NO content in A.belladonna.Therefore,the analysis results showed that the rolB and rolC genes did not increase the TAs content in A.belladonna through H2O2 and NO as signaling molecules.In conclusion,the single or co-introduction of rolB and rolC genes can cause developmental variation in the aerial and underground parts of A.belladonna,but the single introduction of rolB and rolC genes can up-regulate the expression levels of UGT I and H6 H genes and down-regulate the expression levels of TR II genes.Thereby increasing the TAs content of A.belladonna.Although both rolB and rolC genes can increase the content of TAs in A.belladonna,the rolC gene has a more significant effect on the synthesis of TAs in A.belladonna.Therefore,rolC gene is the main rolgene of A.belladonna,and the relationship between rolB and rolC genes is significantly different.Antagonism.The rolB and rolC genes also did not mediate the synthesis of TAs through ROS and NO. |
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