| Maize is one of the world’s highest-yield grain crops and an importantly industrial raw material.In many factors affecting crop yield and quality,photosynthesis is undoubtedly the most influential factor.The amount of chlorophyll in the leaves directly affects the photosynthetic capacity of the plants.The increase of leaf photosynthetic efficiency can make leaves accumulate more photosynthate,plant biomass and economic yield.In addition,many environmental factors also have an important impact on crop yields.Such as trace element zinc,which is associated with many physiological and biochemical reactions in plants.Maize is relatively sensitive to zinc reactions.In the absence of zinc,the chlorophyll content of maize plants decreased,and photosynthesis decreased.In severe cases,maize yield decreased significantly.In this experiment,we used the four transgenic events transformed with zinc-iron transporter gene(ZmZIP1)and the eight transgenic events of the glucose epimerase gene(ZmPHD1)as experimental materials for PCR analysis and real-time Quantitative PCR,Southern blot,Western blot,cytological observations,and investigation of physiological and agronomic traits in order to identify the genetic characters of ZmZIP1 and ZmPHD1 genes,their copy number,m RNA transcription levels,and protein translation levels,effects of genes on physiological and agronomical traits.They will provide a material basis and thereotical foundations for the breeding of high-yield transgenic varieties in maize.The main results are as follow:A total of 12 transgenic events were obtained including 4 transgenic events for ZmZIP1 maize named ZmZIP1-(1-4),and 8 transgenic events for ZmPHD1 maize named ZmPHD1-(1-8).Through PCR detection,the target gene fragments were successfully amplified for each of the events in T0-T4 generations.For T4 generations,1008 plants of ZmZIP1-(1-4)and 1960 plants of ZmPHD1-(1-8)were submitted to PCR assay.As a result,positive transgenic lines without segregation were gained for every event of the ZmZIP1 gene and ZmPHD1 materials,which indicated that target genes were stably inherited across generations.2.In the T4 generations,Southern blot analysis was conducted on 8 transgenic events and 4 transgenic events of ZmZIP1 maize and ZmPHD1 maize respectively.A single hybrid band ranged from 5148 bp to 21226 bp was showed in the transgenic events of ZmZIP1,and no specific band was detected for the negative control(CK).The hybridization results of ZmPHD1-(1-8)showed that all the transgenic events had a single hybrid band,sized in 3530bp-21226 bp,whereas the negative control(CK)had no specific band.Therefore,the target gene was integrated into the maize genome with a single copy,and the insertion sites in different transgenic events were different.3.For the T4 generations,real-time fluorescence quantitative PCR and Western blot analysis were performed on ZmZIP1-(1-4)leaves respectively.The results showed that the relative expression of ZmZIP1 in the leaves of each transgenic event was significantly higher than that of the control(CK),and ZmZIP1-2 and ZmZIP1-3 were extremely significantly higher than that of the control(CK)by 10 folds and 12 folds;Western blot showed that the target protein and PMI protein were both successfully translated in ZmZIP1-(1-4),the protein molecular size was 48 KD and 38 KD respectively,indicating that the ZmZIP1 gene was highly expressed at the transcription and translation levels in transgenic maize lines;and the results of real-time fluorescence quantitative PCR of the leaves of ZmPHD1-(1-8)showed that the relative expression of ZmPHD1 in the leaves of each transgenic event was significantly higher than that of the control(CK)by 5-170 folds,indicating that the ZmPHD1 gene was highly expressed in the leaves of transgenic maize.4.The leaf tissue of T4 ZmPHD1-(1-8)was scanned by transmission electron microscope(TEM)during the grain filling period.Compared with the control(CK),the number of starch grains in the chloroplasts of the transgenic ZmPHD1 maize increased obviously.The results showed that the ZmPHD1 improved the contents of starch in the plants and promoted the synthesis and transformation of carbohydrate in the chloroplast.5.In the T4 generation,the determination of the chlorophyll(SPAD)of ZmZIP1-(1-4)showed that the SPAD of each transgenic event was higher than that of the control(CK)by 3.64%-17.89%.Except for ZmZIP1-4,the difference between the other three transgenic events and the control reached an extremely significant level,indicating that ZmZIP1 promoted the increase of the relative content of chlorophyll in the leaves of maize.6.The chlorophyll relative content(SPAD)and photosynthetic rate of ZmPHD1-(1-8)at the filling stage were measured in the T4 generations.The results showed that comparing with the control(CK),the relative content of chlorophyll(SPAD)of each transgenic increased by 3.69%-16.73%,and under the same light intensity condition,the photosynthesis rate of ZmPHD1 transgenic maize was 18.75% to 42.44% higher than that of the control(CK),and the difference significance test reached an extremely significant difference level.Meanwhile,the light saturation point of each transgenic event was higher than that of the control(CK),indicating that ZmPHD1 promoted the increase of the relative content of chlorophyll in the leaves of maize,and significantly improved the photosynthetic capacity of leaves by increasing the light saturation point.7.For the T4 generations,the phenotypes of all plants of ZmZIP1-(1-4)and ZmPHD1-(1-8)were measured in the grain filling period.The main traits included leaf length and leaf width.For ZmZIP1-(1-4),the leaf area showed the increase of 0.74%-30.99% when compared with the control(CK).For ZmPHD1-(1-8)the leaf area of the transgenic events was higher than that of the control(CK)by 9.18%-50.50%.These results indicated that both genes probably enhanced the biological yield by improving development of leafs.8.In the T4 generations,the yield-related traits of ZmZIP1-(1-4)and ZmPHD1-(1-8)were investigated and analyzed.The main characteristics involved ear length,ear diameter,spike weight,and 100 grain weight and grain weight per spike et al.The results showed that compared with the control(CK),the spike length increased by 4.02%-14.93% in ZmZIP1-(1-4)transgenic events.The grain thickness decreased by 13.86%-21.76%,grain weight per ear increased by 23.25%-76.95%,and the other characters were not changed significantly.Compared with the control(CK),the grain length was increased by 6.65%-12.93% and the grain width increased by 1.64%-10.24% in ZmPHD1-(1-8)transgenic lines,the grain thickness decreased by 0.08%-21.65%,grain weight per ear increased by 9.08%-140.94%,and the other characters were not changed significantly.The results showed that ZmZIP1 and ZmPHD1 genes increased maize yield by improving spike length,seed size and kernel weight,while grain thickness was reduced.In summary,ZmZIP1 and ZmPHD1 genes are related to yield traits and these transgenic lines can be used as basic materials for breeding new high-yielding maize varieties. |
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