Application And Analysis Of Ac/DS Transposition System In Brachypodium Distachyon L. And Wheat

As one of the most important food crops in the world,wheat plays an important role in the world food crops,as well as in China's food security system.Wheat breeding research has always been the top priority of scientists at home and abroad.For a long time,the development of wheat functional genomics has been limited due to the complexity of wheat genome and the lack of high-quality reference genome sequences.Therefore,it is an urgent task for breeders to find good research materials and gene functions,and to find good germplasm resources.The most direct and effective way to study and analyze gene function and obtain good research materials is to study mutants.Therefore,it is an important link to construct a rich mutant library for studying gene function and finding good breeding materials.Ac/Ds transposable system has become the first choice to construct mutant library due to its advantages such as small tissue culture workload,random insertion and phenotypic stable inheritance,and has been applied in many plants.Model plant Brachypodium distachyon and wheat close relatives,and also has many advantages of Arabidopsis thaliana genome?small,short growth period,growth conditions simple,pollinate the flowers,etc.?.Therefore,in this paper,we first introduced the heterologous Ac/Ds transposition system into the Brachypodium distachyon,and then applied the transposition system to wheat.The main results were as follows:?1?The heterologous transposable vector pSQ5 was introduced into Brachypodium distachyon by Agrobacterium mediated method,and 3185 stable plants containing only Ds elements were obtained after 4 generations of cyclic screening.In addition,226 plants with only Ds elements were obtained by introducing the system into wheat after a little modification.The results of multiplex PCR and GUS staining showed that the heterotransposition system could work in Brachypodium distachyon and wheat.?2?Transposition frequency and distribution characteristics.The average transposition frequency of Ds transposon was 6.7%in Brachypodium distachyon and 7.7%in wheat.After mapping the flanking sequence of Ds element by inverse PCR,3057 effective sequences were obtained,2301 specific insertion sites were obtained,507 of which were inserted into exon,241 into intron,152 into 5?UTR,127 into 3?UTR,603 near gene region,671 into gene interval region,and inserted into or near gene region 71%of the total,indicating that Ds elements tend to be inserted into the gene region.As the whole wheat genome sequencing has not been completed,only less than 50%of the flanking sequences obtained by the same technology can be successfully mapped after online blast.The primers were designed to compare the sequences that could not be matched,and the results were verified by multiple batches of re amplification.At last,more than 90%of the sequences are real and effective,only 7%of the sequences could not be amplified,indicating that there are still many gap sequences in wheat.?3?Transposon insertion features.Through the analysis of 2301 specific sites,it is found that at the whole genome level,Ds transposons tend to insert into both ends of the chromosome,while the centromere area is less distributed.In addition,the number of insertion sites has a certain relationship with the size of the chromosome?positive correlation?,and the density of Ds transposons is positively correlated with the gene density of the chromosome itself.?4?Transposons tend to interlock.Four lines with more offspring were randomly selected for study.Among them,#E81 had 227 offspring,#F84 had 134 offspring,#B54 had103 offspring,#H87 had 46 offspring.In addition,the initial insertion site?donor site?of#E81,#F84 and#B54 lines was on chromosome 1,and#H87 was on chromosome 2.After NCBI and JGI online blast,it was found that all the progenies of other lines tended to insert into the same chromosome as the donor site,except for the slight deviation in the distribution of#F84 progeny.The insertion rate of#B54 was 44.7%,#E81 was 47.6%,#H87 was 43.5%,and#F84 was 29.1%?the most insertion site was chromosome 3,accounting for 35%?.Further analysis of the three lines with the initial insertion point on chromosome 1 showed that there were insertion hotspots at the length of 30M.?5?Time of transposition.By GUS staining?according to development time:4-5 leaf stage,heading stage and filling stage,random sampling,5 batches in total,at least 10 plants in each batch,including roots,stems,leaves and various parts of young species?,general PCR and multiplex PCR,In Brachypodium distachyon and wheat,it was confirmed that Ds transposition occurred mainly in the late stage of plant development,from flowering to seed filling.?6?Transposition in wheat.In order to study the specific work of heterozygous transposition system in wheat,Ac and Ds dual transposition vectors constructed on different T-DNA were introduced into wheat?Fielder?,respectively.A total of 139 F₁ lines containing Ac and Ds elements were produced in 29 crosses.A total of 142 F₂ lines were randomly selected from F₁ progeny,and 49 lines?34.5%?were transposed.There are 2283 plants in F₂generation,175 plants containing Ds-only,accounting for 7.7%of the total.Among the lines with transposition,the transposition frequency of different F₂ generations of the same hybrid parent is quite different,which may be caused by different transposition periods of Ds elements.In the same Ac,different Ds parent combinations,the transposition frequency of offspring is quite different,which proves that the initial position of Ds has a great influence on transposition frequency.In the same Ds and different Ac parent combinations,the transposition frequency of the progeny is only different from that of the individual lines.It is proved by different test methods that the transposition frequency of the progeny of the Ac gene with different expression amount is not different.Again,it is the initial position of the Ds element that has great influence on the transposition frequency.?7?Construction of Ac/Ds transposition vectors controlled by specific promoters.Because of the unstable inheritance of somatic transposition,it increases the unnecessary work.Therefore,in order to reduce the trouble caused by this process and improve the work efficiency,the Ac/Ds transposition system controlled by germ cell specific promoter was constructed.The promoter of tomato LAT52 gene was selected as pollen specific promoter,and the successful vector name was pHAD1;the promoter of Arabidopsis thaliana gene Ec1.2 was selected as egg cell specific promoter,and the vector name was pOXQ1.At the same time,we constructed the corresponding promoter to control GUS,named pHG?pollen specific?and pLG?egg specific?respectively.Through Agrobacterium mediated genetic transformation,the constructed vectors were respectively introduced into the Brachypodium distachyon.A total of 136 plants were obtained,including 65 pHG,21 pLG,22 pHAD1 and28 pOXQ1.It was confirmed by GUS staining,multiplex PCR and EDS-PCR that the Ac/Ds transposition system controlled by specific promoter could be successfully transposed in Brachypodium distachyon.?8?The crossing of Brachypodium distachyon.In order to understand the relationship between Ds transposon and target gene,we randomly selected some lines with only Ac element,and selected some lines with only Ds element whose specific insertion site is known,and carried out the hybridization of Brachypodium distachyon?no relevant work has been reported?,and summarized the operation essentials of this method.Among them,the selection of pollination period,the operation methods of emasculation and pollination,and the moisture preservation during the seed development after hybridization are the key to the whole hybridization process.This paper describes each process in detail.