Silencing Of Sgt Gene Family Members Affect Accumulation Of GAs In Potato Tuber

Glycoside alkaloids(GAs)have the effect on inhibiting pathogenic microbial invasion,avoiding insect feeding,osmotic stress and allelopathic effects,but excessive accumulation of potatoes in Solanum tuber affects food flavor and safety.Therefore,it is essential to specifically reduce the accumulation of GAs in the tubers by molecular control,which is very important to improve the resistance of potato and safety of food.The terminal enzyme of the GAs pathway is the three members of the Solanidine glycosyltransferase(Sgt)family(Sgt1,Sgt2 and Sgt3),their expression levels and activity affect the rate and level of GAs synthesis.At present,the regulation of GAs biosynthesis in potato tubers,only one of the members of the Sgt family was studied.But it is very difficult to eliminate the accumulation of GAs in tuber by manipulating the expression of a single gene in sgt gene family.Therefore,in this study,RNAi vectors which were driven by tuber-specific promoter Patatin,was constructed by RNAi technique and used the GAs synthetase pathway terminal enzyme gene family as the target.And two potato genotypes were transformed by Agrobacterium tumefaciens to identify the effects of Sgt gene families on the accumulation of GAs in tubers.This provide a theoretical basis and reference for the use of molecular control techniques to cultivate potato germplasm with low GAs content in tuber.The results of this study are as follows:1.The three terminal enzyme genes of GAs synthesis pathway were searched by bioinformatics method,and the sensitive target location were found.Three gene sensitive fragments were fused by overlapping PCR technique to obtain forward fragment F and reverse fragment R.Then the two fragments were inserted into the intron of the p HANNIBAL vector,and the recombinant plasmid p HAI-FR was obtained.After,the Patatin promoter was used to replace the Ca MV 35 s promoter carried by p HANNIBAL.Finally,the "Patatin P-forward fusion fragment-pdk-reverse fusion fragment-OCS-ter" was digested into the vector p CEPSPS,and the plant expression vector p CEI-PFR containing the ABC reverse repeats of the fusion gene was obtained.And the obtained plant expression vector was integrated into Agrobacterium tumefaciens LBA4404 by freeze-thaw conversion method.2.The genetic transformation experiments were carried out on the potato no axillary bud stems of Zhuangshu No.3 and Favorita by Agrobacterium tumefaciens.After the resistance of glyphosate selection and PCR detection,there were 10 strains(Zhuangshu No.3)and 11 strains(Favorita)with positive expression of reverse repeat transcribed fragments.The positive rate was 0.50%-0.90 %(Zhuangshu No.3)and 0.75 %-0.90 %(Favorita).3.The expression of the genes in the transformed plants was decreased by RT-q PCR.As a result,the expression of three Sgt genes in the transformed tubers was reduced by32%-60%(Sgt1)、 29%-55%(Sgt2)and 25%-66%(Sgt3)respectively,while the expression of glyphosate gene increased by 48%-135%.But the expression of these genes in leaves did not change significantly.4.The content of potato GAs was determined by spectrophotometer.Compared with the wild potato,the GAs content in underground part of the transgenic plant decreased by 41%-59%(Zhuangshu No.3)and 36%-62 %(Favorita),while the GAs content in the aerial part was not significantly reduced.