| Potato(Solanum tuberosum L.)and cassava(Manihot esculenta Crantz)are the important tuber crops,the starch content in cassava root tubers is the highest,in contrast,in potato tubers it is lower.It is very important to cultivate new potato lines which has higher starch content.AGPase is a rate-limiting enzyme in the starch synthesis pathway,it catalyze Glc-1-P to produce ADPG which is a substrate of starch synthesis pathway.Cassava has outstanding characteristics like higher photosynthetic efficiency and starch accumulation efficiency.It is hopefully to improve the efficiency of starch synthesis in potato tubers by transfer the cassava AGPase genes into potato.The AGPase gene family of cassava was analyzed in our laboratory,the Yeast Two-hybrid experiment showed that MeAGPS1a interacted with MeAGPLla and MeAGPL3.We had obtained potato tubers with transferred MeAGPS1a and MeAGPL3 respectively.On the basis of previous studies,identified the traits of univalent transgenic potato tubers.The genetic transformation,molecular identification and trait identification of bivalent transgenic potato tubers.In this study,MeAGPSla,MeAGPLla and MeAGPL3 genes were selected and overexpressed in potato mediated by Agrobacterium tumefaciens method,we expect to get higher starch content of potato lines and identify of its biological function.The research results are as follows:1.In the early stage,potato tubers of 10 lines with transferred MeAGPS1a or MeAGPL3 gene were obtained.After 2 times of vegetative propagation,character identification was carried out.The dry matter rate and starch content of 10 lines were lower than wide type or no difference,the yield of univalent transgenic lines was different.The average yield(Y),average dry matter yield(DY)and average starch yield(SY)per plant for line S-6 was 1.7 times to 2.1 times higher than WT,reach very significant levels;these yield data of line L5 was 1.5 times higher than WT,reach very significant levels;these yield data of line L3 and line L7 were 1.2 times to 1.3 times higher than WT,reach significant level.2.Expression vectors contained MeAGPS1a+MeAGPL1a or MeAGPS1a+MeAGPL3 genes was transferred into potato NO.3 of Hubei by Agrobacterium-mediated.There are 35 positive lines of MeAGPS1a+MeAGPL1a and 43 positive lines of MeAGPS1a+MeAGPL3 by DNA detection respectively.3.There were 10 positive lines which were validated by RNA detection,and showed that these exogenous genes expressed in potato leaves and tubers.Seven lines with relatively high expression level were selected to next step,including 3 lines with MeAGPS1a+MeAGPL1a and 4 lines with MeAGPS1a+MeAGPL3.4.The plantlets of 7 transgenic lines and wild type were propagated and hardening-seedling.Fifteen seedlings of each line were selected to rejuvenation in phytotron,and the temperature and humidity were gradually controlled to external environment.About 121 seedlings were transplanted to a semi-open experimental shed and harvested potato tubers after 110 days.The survival rate of 6 lines exceeded 93%in this process.5.The character identification of 7 lines with bivalent transgenic potato tubers were tested.Dry matter rate and starch content of bivalent transgenic potato tubers were significantly lower than that of WT except for SL3-10,the dry matter rate and starch content of line SL3-10 is 1.5%and 1.0%higher than WT(SL3:MeAGPS1a+MeAGPL3)respectively.The data Y,DY and SY of line SL1-4 were 1.3 to 1.4 times higher than WT,reach significant level.Average number of potato tubers per plant of line SL3-10 and line SL3-12 were 1.8 times and 1.6 times higher than WT respectively,reach very significant level.Three yield data(Y,DY and SY)of line SL3-12 were 2.5 to 2.7 times higher than WT,reach very significant level.This study aims to comprehensively evaluate the characters of WT,univalent transgenic and bivalent transgenic potato tubers.However,the bivalent transgenic potato tubers obtained in this experiment are modren generation,and it needs more than 2 times of vegetative propagation to obtain stable traits.Therefore,further field experiments are required. |
