Construction Of A High Efficient Transgenic System And Establishment Of Versatile Ac/Ds Based Activation Tagging Mutant Generating System In Maize

As an important crop,maize plays a key role in national economic development.Transgenic technology has many advantages comparing to traditional method,such as shortening breeding period,breaking genetic linkage,getting exotic gene easily and so on.So it is necessary to initiate the maize transgenic research.Although many improvements have been made to increase the transformation methods and efficiency of maize in the past twenty years,there are still plant species or genotypes which are recalcitrant for transformation.Agrobacterium mediated transformation has been applied extensively in plant genetic improvement,but the successful reports on maize are limited.It is of great interest to develop new technologies to further improve transformation efficiency in plants.The selection marker could improve the transgenic efficiency,is one of the most critical parts of transgenic system.Effective screening of the transformed cells has been an important part of the development of efficient transformation system.In order to make transgenic selection at the same time to avoid or solve the selection marker had a negative impact,the strategy selection marker and marker relative safety deleting new technology,provides an effective way for the application of the safety of GM,has become an important direction of the development of modern biotechnology.In the post-sequencing era,analysis of genetic mutations is believed be one of the most effective ways to investigate gene function.With the completion of maize genome sequencing,functional genomics aiming at decipherment of function for every maize gene rises and develops rapidly.Large-scale mutant library is an important technique platform for maize functional genomic research,and particularly the activation tag mutant populations are the major part of the platform.In the study,the method of Agrobacterium mediated transformation and new transformation vectors had been adopted to construction of versatile Ac/Ds based activation tag mutant generating system in maize.Main results were as follows:1.Maize inbred line A188 was used as recipient material to establish Agrobacterium mediated embryogenic callus transformation system and obtain insect resistant maize.The conversion efficiency of 38.33% was obtained by the transformation method selection,identification of callus characteristics and the optimization of enzyme pretreatment.Totally,211 transgenic plants from 37 independent transformation events were regenerated by screening of herbicide resistance.Through the PCR,Southern blotting and RT-PCR detection of target gene cry1C*,11 transgenic materials with stable expression of target gene were obtained.ELISA detection and field phenotype test showed that the material had good resistance to corn borer.2.Constructed a new transgenic vector p HZM1N-Rsc and validated by Agrobacterium mediated transformation.In this study,we constructed a transgenic vector,which contains the visual screening marker and an auto-eliminated selection marker-free system.Aiming at this purpose,transformed the vector into maize embryogenic callus by Agrobacterium mediated transformation,the positive transgenic calli were selected using green fluorescent(egfp)as selection marker gene.PCR and Southern blot analysis showed that the green fluorescence screening efficiency was 91.367%.After heat shock treatment,plants of 4 events were obtained and transgenic markers were removed completely.The phenotypic effect,screening and molecular detection of IPTII gene showed that the selectable marker gene was eliminated completely.Transformed transgenic plants without screening markers verified the Rsc function of the target gene through kernel color phenotype.3.Constructed an activation tagged transposon vector p HZM1-Rsc-Activation and transformed into maize to establish an activated tagging artificial transposon Ds system by Agrobacterium mediated transformation.The transformation vector p HZM1-Rsc-Activation was constructed by synthesized an artificial Ds transposon which containing activation tag sequence and plasmid rescue elements and integrated into the Rsc gene on the transformation vector p HZM1N-Rsc.Totally 48 transgenic maize plants from independent transformation events,based on the transformation platform established in this research,which are transposon activation tagging Ds lines. The flanking sequences were isolated from seven activation tag Ds lines by Tail-PCR,which were localized on 1,3,6,7,9 and 10 chromosomes,respectively.4.Bred an Ac line(Zm Ac-B73),which contains a non-active transposon(im-Ac)by means of MAS.Obtained the material Zm Ac-B73-57,which contains a non-active transposon through the analysis of the background recovery rate,the field phenotype identification and the activity of the transposition.5.Obtained 7 activation tag mutants generating lines by crossing Zm Ac-B73-57 and Ds lines.Got the transposition frequency is between 16.5% and 78.3%.Constructed a versatile Ac/Ds based activation tag mutant generating system in maize,implements the corn activation label mutant library building needs.Using an activation tag mutant generating line(DT1),we investigated the distribution of the artificial transposons across the whole genome,breeding the activation tag mutants and selecting the potential mutants.A series of mutants were found deficient in plant height,leaf color,virtual lesion by small-scale screening and 9 mutants were preliminiaryly identified as stable materials with traits with stable genetic characteristics of activation tagged mutant materials were preliminary validation in offspring.