| Maize is an important C4 photosynthesis product and an important feed,industrial raw material and food crop in China.PEPCK is an important metabolic regulatory enzyme in flowering plants and a key enzyme in the C4 photosynthetic pathway of C4 plants.ZmPCK1gene is an important gene in the C4 photosynthetic pathway in maize PEPCK.In this study,the CRISPR vector,RNAi vector and overexpression vector carrying ZmPCK1 gene were transferred into the maize inbred line H99 by Agrobacterium-mediated method,and the transgenic plants with three vectors of ZmPCK1 gene were obtained by progeny molecular biology detection.Through the determination of photosynthetic indexes,functional verification,agronomic traits of T3 transgenic positive plants,the regulation and function mechanism of ZmPCK1 gene on maize photosynthesis was preliminarily analyzed,which lays the foundation for the new maize germplasm with high photosynthetic efficiency was created and cultivation of new varieties were obtained to nurturing lays the foundation.The experimental results are as follows:1.The optimal conditions for genetic transformation of maize callus were studied.The effects of various factors on the yield of resistant callus were as follows:bacterial concentration>co-culture time>infestation time>As concentration.The optimal combinations of resistance callus yield were A2(OD600=0.5),B3（250μmol/L）,C1（30 min）,D2（3d）.2.Agrobacterium-mediated transformation of maize inbred line H99 was carried out by resistance screening,PCR、Southern blotting and SDS-PAGE.5 strains,4 strains and 5 strains of T0 generation positive strains were obtained from CRISPR,RNAi and over-expression vectors respectively.There were 6 strains,5 strains and 5 strains of T1 generation positive strains;6 strains,3 strains and 4 strains of T2 generation positive strains;5 strains,4 strains and4 strains of T3 generation positive strains.The quantitative real-time RT-PCR analysis showed that the positive expression levels of ZmPCK1 in the T2 and T3 generation plants were leaf>cervical>apical,over-expression vector>RNAi vector>CRISPR vector.3.Sequencing analysis of the two mutant plants under T3-3-1 strain of CRISPR vector showed that the sequences of the two mutants were fragmented compared to the untransformed plants,and the sequences of the two mutants were sequenced.The deletion fragments were the same,and there was a mutation in the AGCTCGGCG base deletion at the target sequence position.4.The analysis of net photosynthetic rate of T3 generation positive plants at different time points showed that at 14:00,the three plant-positive and untransformed plants reached the maximum value of Pn,and when the light intensity was strong at 10:00-14:00,all The Pn values of plants increased with the increase of light intensity.The net photosynthetic rate was over-expression vector plants>untransformed plants>RNAi vector plants>CRISPR vector plants.5.Compared with the net photosynthetic rate of three kinds of vector positive plants,the difference between the over-expression vector and the other two vectors at 10:00-14:00 was extremely significant.Compared with CRISPR and RNAi vectors,the difference between the two was significant at 8:00 and 16:00,and the difference was significant at 10:00-14:00.For PEPCK gene analysis,the Pn value of T3 over-expressed positive plants showed a significant decrease in Pn values,which was close to the Pn value of untransformed plants.6.The leaves,stems and roots of T3 generation positive plant were performed senzyme activity analysis.The activity of PEPCK in different parts of different vectors was changed to over-expression vector>RNAi vector>CRISPR vector,the enzyme activity in different parts of each plant is greater than that of the stem and root.The correlation between enzyme activities and net photosynthetic rate of maize transgenic plants was analyzed.There was a significant positive correlation between the two.The coefficient of determination was 0.8256.7.The analysis of agronomic traits of T3 generation positive plants showed significant differences between T3-9-3-3 and control plant heights.T3-3-1-4 and T3-9-3-1 had significant differences from control panicle length.T3-3-1-2,T3-9-3-3,T3-14-1-3 and T3-14-1-4 had significant differences from control ear diameter.The difference between 100-grain weight and control 100-grain weight was significant difference,and 2 was extremely significant difference. |
