| In the face of a global scarcity of water resources and the increasing soil salinization,drought and salt have already become primary factors limiting crop production worldwide.With the growth of population and the pressing demand of biomass resources,increasing food and cash crops production has become a top priority and it is of important strategic significance to breed salt and drought tolerant crop varieties by biotechnology.Maize(Zea mays L.)is an important food,forage and energy crop as well as an important industrial material in the world.It is sensitive to drought and salt stress,therefore,it is of great importance for the improvement of the drought and salt tolerance of maize to further enhance maize yield and utilize the arid and saline-alkali soils.Traditional maize breeding is usually time-consuming, laborious and obstructed by the lack of germplasm resources,while genetic engineering can now be used as a relatively fast and precise means to improve stress tolerance of maize.However,the study of the maize drought and salt tolerance genetic engineering is just at the beginning stage,and it will be an important way for maize breeding to create new varieties with enhanced drought and salt tolerance by genetic engineering in the future.Along with the extensive research of plant genetic engineering,the use of selectable marker genes encoding antibiotic or herbicide resistance for transgenic crops has potentially negative effects.They may cause environmental and consumers concerns that delaying the market process of transgenic crops,and preclude the use of the same marker gene for the selection of multi-transformed plants.Thus,a series of strategies have been developed to either avoid or get rid of 'problematic' selectable marker genes before transgenic plants are introduced into the field.One way to get the marker-free transgenic plants is to excise the selectable marker gene out of the integrated transgene after successful selection.In the FLP/frt site-specific system of the 2μm plasmid of Saccharomyces cerevisiae,the FLP enzyme is a 48kDa protein that covalently binds as four monomeric units to two frt sites and catalyzes the cleavage and ligation of these sites.FLP-mediated DNA excision or inversion occurs depending on the orientation of the frt sites and when they are in the same molecule; the FLP recombinase catalyzes the integration of two DNA molecules when the frt sites are in two different molecules.By controlled expression of the FLP recombinase and specific allocation of the frt sites within transgenic constructs,the system can be applied to eliminate the selectable marker gene from the nuclear genome of the transgenic plants after selection.Based on the problem proposed above,in this work,the elimination of the selectable marker gene als from the genome of the transgenic maize by the FLP/frt site-specific recombination system after successful selection of the transgenic plants (cells)was investigated,and the eliminated frequencies and the ways to increase the frequency were also studied.Alternatively,this study created valuable marker-free transgenic maize materials with improved drought- and salt-tolerance,and made contributions to maize production and the utilization of large area of arid and saline-alkali soils.Furthermore,the study provided a series of technologies to obtain dozens of transgenic maize without marker genes.The main results of this research were summarized as follows:The construction and the application of the FLP/frt site-specific recombination system fitting for monocotyledonous plantsIn this study,the site-directed recombination system fitting for the monocotyledonous transgenic plants to eliminate the selectable marker genes was constructed after a series of procedures,which included the FLP/frt site-specific recombination system fitting for the monocotyledonous plants and the heat-shock inducible FLP/frt site-specific recombination system.To enhance the efficiency of this system,the plant optimal translational modification sequence AACA was immediately adjacent upstream of the ATG initiation codon of the FLP gene,and one full-length frt site and one modified frt site composed of only core recombination sequence were introduced to flank the als gene in directly orientation.In the FLP/frt site-specific recombination system fitting for the monocotyledonous plants,the interest gene AtNHX1 was promoted by the promoter of the rice actin gene;the TsVP and FLP genes were promoted by the promoter of maize ubiquitin gene for their efficient expression in transgenic maize.Aiming at higher elimination frequency of the selectable marker genes in the transgenic maize,and less damage of the overexpression and long-time expression of the FLP recombinase on transgenic plant, the heat-shock inducible FLP/frt site-specific recombination system was constructed. In this system,the FLP recombination gene was under the control of the hsp promoter, and its expression could be induced by the heat shock at specific stages.In the work,FLP recombinase gene,AtNHX1 and als genes,TsVP and als genes were transferred into elite maize inbred lines,respectively,mediated by Agrobacterium turnefaciens LBA4404.After successive self-pollination for several generations,the stably homogenous transgenic lines were obtained.Then the cross-pollination was conducted between these lines either expressing the recombinase gene FLP or harboring recognition sites frt flanked selectable marker gene als to enable excision events.In the hybrids,the frequency of marker gene elimination was 40.35%,and the elimination frequency of the crosses of FLP×frt was slightly higher than that of the crosses of frt×FLP,and the state of completely excised marker sequence was stably inherited.Then the stability of the FLP recombinase of the F1 hybrids was studied.After sexual crossing between the F1 hybrids with FLP and the frt containing transgenic lines,the FLP recombinase gene in the progeny was still expressed and catalyzed the site-specific recombination.When two pairs of frt which flanked their own als gene were introduced into one cell,the frequency of the thorough elimination was decreased greatly,and about 20% progenies without als gene were detected. To determine the efficiency of FLP-mediated recombination when the FLP gene was under the control of the hsp promoter,the homozygous transgenic plants with AtNHX1 and als were cross-pollinated by using the transgenic plants with hsp-FLP. The recombination of the marker excision was induced in two ways,one was the young ear heat shock in situ and the other was the immature embryo heat shock in vitro.In the former way,the young ears were heat shocked at different time after pollination.When the heat-shock treatment was performed at 42℃for 1h after pollination for 24h,the elimination frequency of 60.36%reached the highest.In the later,the immature embryos were cultured on the medium and heat shocked at different time.The highest elimination frequency was 50%,of which the heat-shock treatment was performed at 42℃for 1h after cultured on the medium for 24h.Compared with other reports,this work provided a competitive technology system for the improvement of biosafety of the monocotyledonous transgenic plants. Analysis of salt tolerance of marker-free AtNHX1-transgenic maizeNa~+/H~+ antiporters are ubiquitous membrane proteins that play major roles in the cellular pH and Na~+ homeostasis throughout the biological kingdom.The overexpression of the AtNHX1(Arabidopsis tonoplast membrane Na~+/H~+ antiporter) confers the salt-tolerance of many transgenic plants.In this study,the AtNHX1 gene was introduced into elite maize inbred lines mediated by Agrobacterium tumefaciens LBA4404,and the selectable marker gene als was eliminated by the FLP/frt recombination system.Southem blotting analysis showed the foreign gene was integrated into maize genome and transmitted to the progenies stably.RT-PCR analysis indicated the transgene had stably expressed to various extent in different transgenic maize lines.The salt-tolerance analysis of the progeny of the transgenic lines indicated that the marker-free transgenic maize with significantly improved salt-tolerance was obtained.In this study,the AtNHX1-overexpressing maize plants exhibited remarkably higher vacuolar Na~+/H~+ exchange activities than wild-type(WT)ones before and under salt stress conditions,and the transgenic plants showed increased Na~+ absorption rate compared with WT under salinity stress.These results indicated that the functional expression of AtNHX1 gene conferred the transgenic maize plants a higher capacity to sequestrate Na~+ into vacuoles,reducing the toxicity of excessive Na~+ to the cell.Under salt stress,the Na~+ contents in leaves and roots of transgenic maize lines were much higher than those of WT and the difference was significant. Furthermore,the AtNHX1-overexpressing maize accumulated higher concentrations of Cl~- and K~+ in their leaves and roots than the WT.Therefore,the transgenic plants accumulated more cations in cells to benefit the capability of water uptake and maintain the cell turgor under saline conditions,which finally avoided or reduced the damages caused by salt stress.As a consequence,the transgenic plants exhibited higher seed germination percentage,better growth and higher dry weight than the WT ones under salt stress,suggesting improved salt tolerance in AtNHX1-overexpressing plants.This study creates valuable maize materials for maize salt tolerance breeding in China.Analysis of drought resistance of TsVP-overexpressing maizeIn this work,the TsVP gene(vacuolar H~+-pyrophosphatase gene from a dicotyledonous halophyte Thellungiella halophila)was transferred into elite maize inbred lines Luyuan92 and Ye478 by Agrobacterium mediated transformation.PCR amplification and Southern blotting demonstrated the existence of the foreign gene in transformed plants and their progeny.The expression differences of TsVP gene existed in different transgenic lines monitored by RT-PCR.The drought resistance analysis of the progenies of the transgenic plants at the stage of seedling and flowering indicated that this gene could enhance the resistance of transgenic maize to drought stress.Under drought stress,it was shown that the heterologous expression of TsVP gene promoted the growth of transgenic maize root system,heightened total biomass and root/shoot ratio significantly.For instance,after drought stress,the biomass of plant aerial part and root,and the root/shoot ratio of L1 were 31.6%,76.4%and 36.4%,respectively,higher than those of WT-L.These findings indicated that the transgenic plants could modify the growth and development of the organ for water uptake against the limited soil water,resulting in a more favorable plant water status. And the better development of root system in TsVP-overexpressing maize plants may provide the morphological and/or physiological basis for enhanced performance under drought conditions.The measurement of isolated vacuolar membrane vesicles from the TsVP-transgenic and WT plants demonstrated that the transgenic plants had higher V-H~+-PPase activity.And the performance of the transgenic maize in response to osmotic/drought stress was better in those lines that had higher V-H~+-PPase activity. The transgenic plants showed higher percentage of seed germination,higher relative water content,less membrane damage and lipid peroxidation and lower solute potential than the WT plants under osmotic stress.The main reasons for the higher drought resistance of the TsVP-overexpressing maize could be attributed to(ⅰ)a more extensive root system,which facilitated the water uptake from a greater volume of the soil during periods of growth on limited soil water to reduce the extent of plant dehydration,and(ⅱ)the increased capacity to absorb and retain water by lower solute potential,which made the transgenic plants maintain normal cellular turgot,and avoided the damages caused by drought stress.Drought resistance of maize at the stage of reproductive development is crucial for grain yields.To study the effects of drought stress on maize plants at their reproductive stage,both the transgenic and WT plants were subjected to drought stress with a shelter protecting from the rain for 6 weeks beginning at the 10-leaf stage, and the changes of their physiological characteristic were detected at drought stress for 0,1,5,10 and 14d.The results were in agreement with the findings of seedlings under osmotic stress,indicating that the transgenic plants accumulated more solutes in cells,remained higher relative water content in leaves and lowered the membrane injury.Furthermore,the ASI(anthesis-silking interval)of transgenic maize plants was shorter than that of WT.After drought treament the grain yields of transgenic lines were much higher than those of WT and the difference was significant.The 1000-grain weights of L1 and L4 were 27.6%and 23.0%greater than those of WT-L, respectively,and those of the Y1 and Y4 were 15.4%and 19.0%,respectively,greater than those of WT-Y.These results suggested that the beterologous expression of TsVP could effectively improve the drought resistance of the transgenic maize plants.In this work,the TsVP gene was transferred into maize for the first time,and it was illustrated that the H~+-PPase played an important role in maize drought resistance by analysis of the progenies of the transgenic plants under drought stress conditions. Moreover,the transgenic maize plants were obtained with higher grain yields than non-transgenic controls after drought stress,and the valuable maize materials for maize drought resistance breeding were created.In summary,the site-specific recombination system fitting for the monocotyledonous transgenic plants was constructed to eliminate the selectable marker genes with high efficiency and precision.Such a system was applied successfully in transgenic maize,indicating that it was well suited for high frequency marker elimination in cereals,and also laid a foundation for its application in other monocotyledonous transgenic crop plants.Furthermore,based on the mechanism of the site-specific recombination,besides the precise excision,it offered the possibility of directed integration of transgenes at characterized loci and the transgene pyramiding.In addition,our work produced valuable maize materials without the selectable marker gene for maize salt and drought resistance breeding,thereby it had a great potential for the contributions to food security in China.
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