Construction Of RNAi Expression Vector Targeting Gbss, SsⅢ And SsⅡ Gene In Potato And Regeneration Of Transgenic Plants

Potato(Solanum tuberosum L) is an important raw material for starch production.So potato starch has superior trait and have been an important raw material for many industrial applications,such as industry,food service industry and chemical engineering.In the most application domains,the potato starch is requested to possess lower-gelatinization temperature and faintish retrogradation,so they have been the important index.However,the two natures are related to the chain length,the ratio of amylose content to amylopectin and the structure of starch.Therefore,to cultivate a kind of potato with the low gelatinization temperature and faintish retrogradation has important significance for improving the application range of potato starch and the promote the industrial development.In the past few years,with the further reserch of enzemy related to the biosynthesis and molecular mechanism of potato starch,it will be possible to use technique of genetic engineering to regulation the key enzymatic gene expression for improving the content,branching degree and the length of amylopectin.For the above mentioned goals,we do some research and expect to get the new variety of potato which has lower-gelatinization temperature and faintish retrogradation nature by silencing the gbss,ssⅢand ssⅡendogenous genes of potato tuber with RNA interference.The following are the main works we did:(1)The gbss,ssⅢand ssⅡgene sequence homology of potato were analysed with the blast method and molecular biology software DNA Star,the gbss(1～261),ssⅢ(2 164～2 407) and ssⅡ(161～441)fragments were selected from these genes and were taken as target.At the same time,the three acquainted gene fragments were fused by the one-step PCR method,the fused gene we obtained is a 785 bp fragment.(2)In order to construction of RNAi expression vector,the 5' flanking region of gbss gene was sub-cloned from plasmid LS-8 and the tuber-specific-promoter patatin was also sub-cloned from plasmid LS-4,the two kinds of promoter were inserted the restriction site Sac I at the 5' end and the restriction site Xho I at the 3' end.(3)Construction of ihRNAi Expression Vectors of"forward direction gbs3s2-pdk(intro)-reverse gbs3s2"regulating by the 5' flanking region of gbss gene and patatin tissue specificity promotor for breeding a low gelatinization temperature potato.(4)In this study,the item of potato as the explant,Some parameters of Agrobaterium tumefaciens mediated inheritable transfromation was established.Based on the MS medium,the density and the assignment of the hormones was optimized,finally to confirm the optimum medium in favor of the growth of the callus:MS0+1.0 mg/L 6-BA +0.3 mg/L NAA(NHD3);MS0+1.0 mg/L 6-BA +0.2 mg/L NAA (NC);MS0 + 1.0 mg/L 6-BA +0.4 mg/L NAA(NF).(5)The pARTGF was transferred to three fine potato varieties plants by agrobacterium tumefaciens system.the resistance plants were obtained and the results of pcr indicated that 10 regenerated plants were positive,there were 2 NC plants,3 NF plants and 5 NHD3 plants among the total.