| Brachypodium distachyon is a new kind of temperate gramineae model material. It playsan important role, such as food, forage and energy in fields. Contrast to other temperte cerealsBrachypodium distachyon as Arabidopsis thaliana has lots of advantages: genome is small,the small size, rapid generation time, self-fertile, a high efficiency transformation and so on.In recent years, about studies Brachypodium distachyon has achieved rapidly development. Itmainly depends on the two aspects: on the one hand, Brachypodium distachyon（ecotypeBd21）genome sequencing has completed; on the other hand, Agrobacterium-mediatedtransformation has been broken through.The first step get the mutant materials for studying of gene function. So far theArabidopsis and rice genome have been basically achieved the saturation of mutations, ratherthan the work of Brachypodium distachyon mutant materials collection work is still in itsinfancy. The progress has been slow. By now, it has two main ways to get Brachypodiumdistachyon mutant materials: the first way is chemical mutagenesis（EMS mutagenesis）; andthe second way is T-DNA insertion mutation. In2013about50000T-DNA are expected toobtain. The mutant material100000of T-DNA are necessary for saturated mutations.Chemical mutagenesis can quickly obtain a large number of mutants in the early, but genesare difficult to locate. It takes a lot of time to locate the mutant genes. In the opposite, T-DNAinsertional mutagenesis need to spend a lot of labour and capital in the early stage, but in thesubsequent phase it’s very convenient to find T-DNA insertion position for locial relatedgenes in the subsequent phase. Considering the time, labor, capital, etc factors, this paperdescribes the induced Ds transposition system for Brachypodium distachyon efficientapplication.Transposition system have been widely used in Arabidopsis, rice, tobacco and othermaterials. Ac/Ds system is the most widely used transposition system. This system ischaracterized by: Ds biases inserted into the interior gene; and tends to jump and insert in near the original insert location; living the8bases of direct repeat sequences. In theory, as long asthere is Ac-TPase exists, Ds leap is a certain probability jump. It can lead to the original insertunstable Ds. To overcome the shortcomings of Ac/Ds translocation system, this paper use twoinducible Ds transposition systems: ethanol induced Ds system （pHL1）; dexamethasoneinduced Ds system（pHL2）. Under the conditions of ethanol, pHL1can produce Ac-Tpase toprompts the Ds jump. New insertional mutagenesis will been produced. Ac-TPase and GRbelongs to the fusion protein in pHL2vector. Under the condition of exogenous addingdexamethasone, the fusion proteins are able to enter the cell nucleus, and prompted the Dsjump. New insertional mutagenesis will been produced. Through the induced system, we canguarantee the stability of Ds, and can also make a leap for Ds.To the aim of this study, we use induceble Ac/Ds system to build a high density of T-DNA group and quickly get a lot of new insertion mutation in a short period of time byinduced. Thus, the saturated mutations in Brachypodium distachyon genome can be easilyachieved.By Now we have raeceived1000T-DNA lines about pHL1, pHL2and constitutive ofAc/Ds. For pHL1with ethanol induction, it can detect Ac-TPase expression quantity increasessignificantly, and the highest expression quantity have increased more than200times. At thesame time, the background levels of Ac-Tpase show differences among different plants ofpHL1. We also use the method of PCR to detect the work conditions of our inducible system.In pHL13lines were detected Ds transposition from13lines; In pHL2with dexamethasoneprocessing,8lines were detected Ds transposition from10lines and among. Constitutive ofAc/Ds system, semiquantitative PCR results dspaly that nine out of10strains of Dstransposition can be detected. After Ds transposition from pHL1, pHL2and constitutive of Ac/Ds system, at the moment, we obtained31sequencing results for the Ds transposition.Finding that Ds can either take away or inserted part of the bases in the8bases repeatsequences.On the oher hand, in the process of building T-DNA high density insertion group, weobtained a Brassinosteroid synthetic defect line. Through the positioning of T-DNA andphylogetic relationship analysis, we found that it’s a C6-oxidase gene in the brassinosteroidsynthetic way. Complementary tests are carried out using brassinosteroid C6-oxidase genome length. And the plant recovered to the wild type as we expected. At the same time thehomozygous materials are also treated by exogenous BR. It is part of the back to the wild type.This result furtherly confirmed that it is a loss of BR mutant. Acording the results of scanningelectron microscopy and tissue section, the cell morphology of mutant materials have changedsignificantly. The transcript level for brassinosteroid C6-oxidase was assessed in differentplant tissues using qRT-PCR. The C6-oxidase gene can be detected in all tissues assessed, butshowed significant variation in transcript levels. The level of expression in the sheath tissuewas the highest. And the promoter of the C6-oxidase gene driving report gene GUS stainingresults showed that the color is the darkest in leaf sheath. |
PDF Full Text Request