| Achieving high yield of crops is a crucial goal of crop breeding.As the major crop in the world,the improvement of yield trait of maize（Zea mays L.）is related to food security closely.Photosynthesis plays a decisive role in the formation of crop yield.Especially the chlorophyll content in plant leaves directly affects the photosynthetic efficiency.In addition,the formation of crop yield is regulated by many trace elements,such as zinc,which is involved in the synthesis of many key enzymes in plants.Therefore,it is important to improve elite inbred lines and create new breeding materials by making good use of gene resources which controlling photosynthetic efficiency and zinc transport in breeding practice.In the previous study,we obtained the 4 transgenic events transformed with the zinc-iron transport gene（Zm ZIP1）and the 8 transgenic events transformed with the glucose epimerase gene（Zm PHD1）both in their T2generations,named them Zm ZIP1-（1-4）and Zm PHD1-（1-8）,respectively,and noted briefly in Z1-Z4 and P1-P8.In this study,T2 positive（contain the target gene）plants were used as donor of those transformed events,7 elite inbreed lines:18-599,RP125,RP128,LH8012,YA8201,YU561,and R08 which generalize mainly and still with great utilization potentiality in Southwest China were used to create near-isogenic lines as recipient materials and recurrent parents respectively.During the whole process of material creation,specific PCR detection and genetic stability verification of target gene were carried out in each or early generations.The related agronomic traits and related ear yield traits of F1hybrid combinations were investigated and then conduct phenotypic difference analyzation between the positive plants（contain target gene）and negative（contain target gene）plants.Southern Blot and Western Blot detection experiments were carried out to identify the Zm ZIP1 gene and the Zm PHD1 gene in their gene heredity condition,copy number,and target protein expression of F1 hybrid combinations simultaneously.Adopting the 40 SSR primers in the protocol for the identification of maize varieties of SSR maker method,which from the standard of agriculture industry in our country to detect the background revert rate,combine with identification of the field phynotypes to filtrate the material.The results of this study were as follows:1.A total of 65 F1 hybrid combinations were compounded by hybridizating 12transformation events in T2generation and 7 maize elite inbred lines.The agronomic and ear yield traits of F1 hybrid combinations were investigated,and the improvement of those traits was evaluated.There come out that 5 hybrid combinations significantly heigher than that of the control in plant height which harboring Zm PHD1 gene and the same number as that as harboring Zm ZIP1 gene.14 hybrid combinations significantly heigher than that of the control in ear height which harboring Zm PHD1 gene and 11hybrid combinations significantly heigher than that of the control in ear height which harboring Zm ZIP1 gene.The hybrid combinations significantly increased in both leaf length and leaf width are R08×P4,R08×Z1,LH8012×Z1,RP128×Z4,and YU561×Z1.The number of combinations of ear length,ear diameter and ear weight and grain weight per ear was significantly higher than that of the control are 4、0、3、3 in harboring Zm PHD1 gene,and 4、2、3、3 in harboring Zm ZIP1 gene,respectively.The traits ear length,ear diameter,ear weight,and grain weight per ear of hybrid combination RP125×Z4 were all significantly higher than those in the control,suggesting that it have greater application potential.2.Selected F1 hybrid combinations that both containing 8 Zm PHD1 transgenic events and the elite inbred lines randomly to do Southern Blot analysis of maker gene<sub></sup>PMI.The results showed that the exogenous T-DNA sequence integrate into the corresponding F1 combinations in single copy form.The hybridization bands were between 2027 k D-21226 k D and vary from each other,revealed that different insert positions of exogenous genes in genomes in different transgenic events.The similar result gained when do the same analyzation selecting the F1 hybrid combinations contained the 4 Zm ZIP1 transgenic events and the hybridization bands is vary from5148 k D-21226 k D.3.Western Blot detection was performed on the same F1 hybrid combinations which selected in Southern Blot analysis in result 2.The results showed that the target protein or the marker protein PMI were expressed,but the brightness of hybridization bands among different combinations was significantly different,confirmed the valid expression of Zm PHD1 and Zm ZIP1 in the hybrid combinations,and different protein expression levels in hybrid combinations prepared by different transgenic events.The expression of marker proteins PMI in hybrid combinations prepared by Zm PHD1-2,Zm PHD1-3,and Zm PHD1-5 which transformed with Zm PHD1 was higher than other combinations prepared by other events,and the molecular weight is 48 k D approximately,in line with the expectation.The expression of target proteins in hybrid combinations prepared by Zm ZIP1-2,Zm ZIP1-3 which transformed with Zm ZIP1 gene was higher than other combinations prepared by other events and Zm ZIP1-3 is the highest in marker proteins PMI expressing than oher events.The molecular weight of the maker protein PMI was about 48 k D,and the molecular weight of the Zm ZIP1 target protein was around 30 kd,both in line with expectation.4.BC1F1,BC2F1,BC3F1,BC4F1,BC2F2,and BC2F3 backcross generations were prepared.Specificity testing was applied to detect the import gene in each generation and verified the sequence of exogenous target genes.Chi-square test was also conducted for the number of positiveand negative plants in offspring of BC1F1 and BC2F1 materials.The results showed that most combinations conformed to be the Mendelian segregation rule of trait heredity controlling by one pair dominant allele.Combine with selection of phenotypic traits to create near-isogenic line materials simultaneously.5.In the backcross generation BC2F2,adopting 40 primers in SSR marker method according to the Agricultural Industry Standard NY/T1432-2014 to identify maize varieties in our country to detect and identify background revert rate.Twenty-two backcross combinations with 97.5%（only 0 pair or 1 pair of primer differed）background revert rates were successfully selected,accounting for nearly 50%of the total combinations.Field phenotypic identification of these background homozygous transgenic backcross materials showed that their plant-type-related traits were basically identical with those of their recurrent parents,which demonstrated that background-based molecular marker-assisted selection could improve the accuracy of selection and shorten the duration of transgenic breeding process.In future,the yield traits of these homozygous transgenic backcross materials will be identified,hoping to filtrate and breed the greater elite maize inbred lines. |
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