Construction Of Ac/Ds Transposition System In Maize(Zea Mays L.) And Preliminary Analysis Of The Function Of BdSOC1-like Gene In Brachypodium Distachyon L.

Maze(Zea mays L.)is one of the most widely distributed crops in the world.It is widely used as a human ration and can also be used as a livestock Feed and of raw materials of industrial production.As the proportion of meat in the residents' dietary structure gradually increased,the development of the aquaculture industry was greatly promoted.With the rapid development of aquaculture,the demand for feed has risen year by year.Corn can also be used for industrial raw materials,the production of starch,amino acids and ethanol,which led to a steady increase in corn acreage and total output.But corn demand is still growing in the world,and the supply situation is not optimistic.Therefore,it is very necessary and urgent to actively explore the molecular genetics research of maize and provide material and genetic resources for maize genetic breeding.However,the study of molecular genetics of maize is inseparable from transgene and a large number of mutant materials.In addition,the large plant type of maize is not suitable for large-scale greenhouse growth,and the growth cycle is long,which limit the development of maize gene function research.Brachypodium distachyon is closely related to barley,wheat,corn and other gramineous crops,all of which are monocotyledons.In addition,Brachypodium has small genome,short growth cycle,simple growth conditions and self-pollination,which make it a model material for studying gramineous monocotyledons.In order to better carry out functional genomics research on maize.This paper is devoted to the establishment of efficient and reproducible maize genetic transformation systems and Ac/Ds tag mutant library.A large number of mutant materials were created to lay the foundation and provide materials for the exploration,functional annotation and molecular mechanism research of maize genes.At the same time,we also carried out a preliminary analysis on the function of the relevant mutants and related genes in Brachypodium,so as to provide ideas for the research on the molecular and evolutionary relationship of gene function among various species.The establishment of maize genetic transformation system(1)The maize transformation through particle bombardmentDuring the genetic transformation of maize,the immature embryos or calli from them were used frequently as recipients for transformation and endured the prolonged selection by antibiotics or herbicides,to produce resistant calli and shoots.Due to the fact that escapes are quite common from the selection by antibiotics or herbicides in maize transformation,additional jobs are required to deal with this.Using a plasmid containing GFP(Green Fluorescence Protein)gene as a visual marker,the calli of immature embryos of maize inbreds A188 and Qi319 were bombarded.In a transient expression experiment,the GFP fluorescence was observed within 3w and kept at a high level in 10 d after bombardment.Under the selection of hygromycin and by the visual check of GFP fluorescence,a few transgenic maize plants were produced with a frequency of 0.3-0.5%.The identity of the transgenics was confirmed by the molecular analysis assays and genetic analysis experiments of offspring later.This work suggests that GFP is a good visual marker to pick up the real transformants from the resistant calli and shoots of maize.(2)Agrobacterium-mediated maize transformation In Agrobacterium-mediated transformation research of maize immature embryos(IEs),the MS or N6-based cocultivation medium(MC)was routinely preferred culturing the IEs.In our studies,here,a novel co-culture method-Dry filter paper co-culture method(the immature embryos inoculated with Agrobacterium harboring target vector were placed on two filter papers in a petri dish,DC)is described in the Agrobacterium–mediated transformation of maize Qi319 immature embryos.To examine the effect of the two co-culture methods(MC and DC)on transformation efficiency,we designed three experiments:(1)A.tumefaciens strain AGL1 independently carrying two plasmids,pXQD12 and pXQD70;(2)two A.tumefaciens strains,AGL1 and EHA105,carrying pXQD12;and(3)strains AGL1 and EHA105 each independently inoculated with pXQD12 and pXQD70 for different infiltration periods,5,10,15,20 and 25 min.We used A.tumefaciens to inoculate IEs derived from maize ears 9–15 d after pollination,and then IEs were placed in petri dishes for co-culturing.The DC treatment significantly increased the percentage of IEs expressing green fluorescence protein(%GFP),indicating positive transformants.DC-treated IEs had ~3-4 times the %GFP compared with MC-treated IEs at 8 d after inoculation(3 d co-culture and 5 d restoration).The average regeneration frequency(%GFP positive regenerated calli of infected IEs)and stable transformation frequency(%GFP positive T0 plants of infected IEs)significantly increased with the DC treatment.Thus,the DC method may be used to develop a more efficient Agrobacterium-mediated transformation method for maize IEs.Construction and analysis of an Ac/Ds tagging mutant library in maizeMaize is a model organism for C4 Plant in both molecular and agronomic research.This project utilized the transposon-tagging construct Activator(Ac)/Dissociator(Ds)-ATag_ubiGFP to produce insertion-tagged and knockout mutants in the processing maize Qi319.The construct carried neomycin phosphotransferase(NPTII),green fluorescent protein(GFP),and the transposase(TPase)of maize Activator major transcript X054214.1 on the stable Ac element,along with a 35 S promoter in vitro propagation strategy was used to produce a population of 4 T0 plants in which Exogenous Ds(fDs)coud jump from one site to another site.A population of 12,514 self Qi319 backcrossed progeny was produced from the functional T0 line and T1 line.This population was screened using green fluorescent protein(GFP)and multiplex polymerase chain reaction(PCR).Insertion sites of transposed Ds-ATag elements were identified through thermal asymmetric interlaced PCR(TAIL-PCR)or inverse PCR(iPCR),and resulting product sequences were aligned to the recently published maize genome.A population of 154 independent,fDs only transposant lines spanning all 10 maize chromosomes has been developed.The transposon-tagged lines have been immortalized in seed stocks and can be accessed through an online database,providing a unique resource for breeding and analysis of gene function in the background of a commercial maize cultivar.The preliminary study on the function of BdSOC1-like gene in BrachypodiumIn the above Ac/Ds mutant library,we found a late flowering mutant possibly caused by fD insertion.Through molecular and bioinformatics analysis,fDs is inserted into the first exon of maize gene whose gene number is NP001105152,and the gene was homologous with BdSOC1-like gene in Brachypodium.Because maize growth period is longer,larger plant type is not suitable for greenhouse on a large scale growth and other characteristics,in order to quickly research the function of NP001105152 maize genes,at the same time,we also want to see the functionality of the homologous genes in temperate grasses,the function of BdSOC1-like was preliminarily analyzed by using Bd21,a model plant closely related to wheat and rice and with short growth cycle.In this study,Bioinformatics analysis suggested the presence of a conservative MADS-box and keratin(K)domain that belong to the SOC1 family.The expression of Brachypodium SOC1-like(BdSOC1-like)was widely detected in different tissues/organs of Brachypodium,including roots,stems,young leaves,old leaves,shoot apical meristem(SAM),and flowers at different developmental stage.Also,quantitative real-time(qRT)PCR results suggested that the accumulation of BdSOC1-like gene did not display a diurnal rhythm under long-day(LD,18-h light/6-h dark)or short-day conditions(SD,10-h light/14-h dark),and its expression did not appear to remember the prolonged cold treatment in Brachypodium.The heading date was earlier in plants overexpressing BdSOC1-like,which led to a reduced seed set in Brachypodium.In combination,above results suggest that BdSOC1-like may play important roles in the transition to flowering in Brachypodium.In conclusion,this study improved the genetic transformation of maize,established the Ac/Ds transposition system,and performed a functional analysis of a flower-related maize homologous gene in Brachypodium.three innovative results were obtained.(1)A method for GFP tag identification was established in particle bombardment-mediated transformation of maize and it make the identification of transgenic offspring faster and more convenient.The dry filter paper co-culturing method was established in the Agrobacterium-mediated transformation of maize to increase the transformation efficiency.(2)The maize Ac/Ds transposition system was established,and 154 fDs transposition lines with transposition locus distributed on 10 chromosomes were obtained.(3)In maize,a late flower mutant was found,and the mutant gene was analyzed.Then the homologous gene BdSOC1-like was found in the genome of Brachypodium,and its function was verified by transgenic method.