Study On Prokaryotic Expression Of AhPPDK Gene In Amaranth Hypochondriacus And Genetic Transformation Of AhPEPC Promoter

Amaranthus hypochondriacus is annual herb plant,belonging to the C4 plant.In this paper,correlation analysis and path coefficient analysis were used to study the factors affecting the net photosynthetic rate;In this study,we cloned the PPDK gene(AhPPDK)(GenBank registration number:JF907701),and the recombinant protein of the prokaryotic expression was obtained and the activity of the enzyme was determined;Agrobacterium tumefaciens mediated transformation was used to identify the AhPEPC promoter with different length of deletion in tobacco and potato.The results are as follows:(1)Amaranthus hypochondriacus net photosynthetic rate(Pn)and temperature(Tm),photosynthetically active radiation(PAR),transpiration rate(Tr),stomatal conductance(Cleaf)correlation analysis showed that Tm,PAR,Tr and Pn were positively correlated,and the correlation reached extremely significant level,negatively correlated with Cleaf,CO2 int and Pn,and the correlation is reached the extremely significant level;the correlation analysis between the factors that,in addition to E and Cleaf were significantly correlated,among other factors have reached very significant level.Path analysis showed that the path coefficient of PAR,Tr,Cleaf and Pn were positive,and the path coefficient of Tm?CO2int and Pn were negative,the relative importance of the 5 factors to the net photosynthetic rate(Pn):PAR(0.339)>Tr(0.299)>Cleaf(0.251)>Tm(-0.522)>CO2int(-0.908).(2)The recombinant plasmid pEASY-El-AhPPDK was transferred into the competent cells of Escherichia coli strain Transetta(DE3),and the 2h,4h and 6h were induced by 1mmol/L IPTG inducer and analyzed by SDS-PAGE gel electrophoresis.The results showed that the recombinant AhPPDK was highly expressed and the molecular weight of the protein was about 108 KDa,which was consistent with the expectation and the expression level of protein was the highest at 6h.After the ultrasonic waveof Escherichia coli was induced by 6h,the supernatant and precipitation were detected by SDS-PAGE electrophoresis.The results showed that the target protein was mainly expressed in the supernatant.The activity of the enzyme was determined by spectrophotometry.The results showed that AhPPDK expressed in Escherichia coli had the activity of pyruvate kinase.(3)Agrobacterium tumefaciens mediated transformation of AhPEPC promoter with different length of deletion into potato and tobacco was carried out.The transgenic tobacco and potato plants with 1800 bp and600bp promoter of AhPEPC were obtained.The results of GUS histochemical staining of transgenic tobacco leaves were analyzed.The results showed that the expression of GUS was increased under light condition than that under dark culture in the leave.