Construction And Application Of Ac/Ds Transposition System In Brachypodium Distachyon L.

Wheat is one of the major grain crops in China and plays a decisive role in the national food security system.For a long time,due to the complexity of the wheat genome and the serious lack of high quality of the whole genome sequences,the inefficient genetic transformation system,the large plant type which was not suitable for large-scale greenhouse growth and long growth cycle,these factors limited the functional genetics of wheat.Brachypodium distachyon belongs to the precocious subfamily and has a close relationship with barley,oats,wheat and other crops.B.distachyon also has a series of similarities advantages with dicotyledonous model plants like Arabidopsis thaliana（small genome,short growth cycle,simple growing conditions,self-pollination,etc.）and has been proposed as a good model plant of temperate poaceae.The work of gene excavation and functional annotation need a large number of mutant materials.There are many ways to create mutant materials,generally divided into natural mutagenesis,physicochemical mutagenesis,T-DNA insertion mutagenesis,and transposon insertion mutagenesis.These methods have their own advantages and disadvantages.Natural and physicochemical mutagenesis can produce mutant material in a short period of time,but it is difficult to find out the genes causing the phenotypes.T-DNA insertion mutagenesis can use known tag sequences to find out the insertion sites easily,but it requires a lot of tissue culture work to enrich mutant materials.Transposon insertion mutagenesis overcomes the shortcomings of the above methods,through constructing a small number of starting individuals,a large number of transposants can be obtained in the offspring and the insertion sites can be identified easily according to the known vector sequences.The mutants produced by transposon tagging can accelerate the analysis of gene function.To better carry out functional genomics research in wheat,this study mainly focused on the following two aspects:On one hand,constructing Brachypodium distachyon high-efficiency Ac/Ds transposition system,creating a large number of mutant materials,studying gene function and its mechanism,and finding wheat homologous genes for analysis,the work will speed up the research process of wheat functional genomics.On the other hand,the high efficient Ac/Ds transposition system of Brachypodium distachyon was transformed into wheat to explore its working efficiency,mutants identified in the Ds tagging population will create good conditions for searching for valuable genes and carrying out gene function and molecular mechanism research.Construction of an efficient Ac/Ds transposition system and mutant library in Brachypodium distachyon L.In this paper,we constructed an efficient Ac/Ds transposition system and enriched a certain scale of transposants in which some mutants related to agronomic traits were screened out.The main results were as follows:（1）We constructed one-element Ac/Ds vector（pYX1）.The transposase gene in the Ac element was driven by Ca MV 35S promoter and NPTII gene as a marker gene was used to identify the existence of Ac element.Green fluorescent protein gene（GFP）driven by the maize ubiquitin 1（ubi1）promoter in the Ds element acted as an efficient positive selected marker to identify transposants.β-glucuronidase gene（GUS）driven by the 2×CaMV 35S promoter（2×35S）will be expressed when the Ds element transposed from the donor site and acted as a positive marker gene to detect somatic transposition.We acquired 477transgenic lines by transforming Brachypodium distachyon cultivar Bd21 through Agrobacterium-mediated method.（2）Early somatic transposition of T₀ lines was detected by GUS staining and PCR amplification and semi-quantitatively confirmed that the Ac transposase gene was expressed.（3）A certain number of transposants were enriched in the T₁ population.The main screening strategy was to conduct G418 antibiotic treatment on large-scale T₁ plants,followed by observing green fluorescence under a microscope,and finally screened out the candidate plants in which transposants will be identified by multiplex-PCR.Experiments showed that the average screening efficiency was about 70.4%,and the transposition frequency in T₁ generation was between 0.00%and 12.50%.（4）So far,we have obtained more than 600 site-specific insertion sites of Ds element by combination of antibiotic treatment and green fluorescence observation.The border sequences of the insertion sites were obtained by Tail-PCR,and then the sequences were BLASTed against the Brachypodium genome assembly on the website,and finally the detailed information of the insertion sites of the Ds element was obtained.By analyzing the localization results,we found that Ds elements preferentially transposed into genic region,and the detailed information were as follows:promoter（22.2%）,extrons（18.7%）,introns（8.7%）,3′UTR（6.5%）,others（43.9%）.Ds elements distributed widely on the five chromosomes of Brachypodium distachyon,but there is less distribution near the centromere.The transposition time of the Ds elements was late.The insertion sites of the transposants（T₁ family）from 36 T₀ lines were analyzed and two or more than two different Ds insertion sites were found among the 32（88.88%）T₀ lines.Ds tended to transpose to closely linked location with the original T-DNA site.Through analysis of the insertion sites of the transposants from T₁-T₄ generations of 18,27 and 140 parental lines,we found that Ds elements distributed on the chromosome closely linked location with the original donor sites.（5）Through phenotypic screening of the Ds insertion populations,we found many mutants about plant height,leaf color,seed size,spike shape,leaf width,germination rate,flowering time,and fertility variation（This article only listed some of the mutants caused by Ds insertion）.Partial of the interested mutants have performed preliminary functional analysis.In this paper,two yellowing mutants were taken as an example,the location of the Ds insertion sites were identified by Tail-PCR.The mutant gene in 18-29-1-28 mutant was a TPR superfamily member gene and was named as TPR like 1（Bd TPRL1）.Quantitative results showed that the expression level of the Bd TPRL1 gene was down-regulated.Experiment analysis indicated that the phenotype was controlled by the Bd TPRL1 gene,homozygous showed yellow leaves and a lethal phenotype（two-leaf stage）.the other mutant was 18-12-6-5-4 in which the mutant gene was predicted as an enzyme involved in pectin synthesis,this gene was named as leaf yellow 1（Bd LY1）,the mutant showed yellow leaves and lower plant height than the wild type.Application of the Ac/Ds transposition system in wheatTo explore the effect of the high efficiency Ac/Ds transposition system of Brachypodium distachyon in wheat,we transformed the pYX1 vector into the wheat variety CB037（Triticum aestivum L.）.It was proved by experiments that the Ac/Ds transposon system could also work in wheat,and Ds only plants were screened out in T₁ population,and the flanking sequences of Ds insertion were successfully separated.The detailed results were as follows:（1）Three transgenic wheat lines were obtained by Agrobacterium-mediated transformation method and named as A1,B1,and C1,respectively.（2）In T₀ generation,Ds elements transposed from the donor site which was confirmed by EDS-PCR（empty donor site）.After Ds transposing from the original location,there remained footprints which caused insertion,deletion or transversion of the Ds flanking sequences.（3）Transposants were screened by green fluorescence observation and PCR reaction in the T₁ generation of B1 plant（T₀）.Flanking sequence was obtained by Tail-PCR and aligned with the public wheat genome,we found that Ds element inserted into the 4AL:32237 locus.