Mutants Screening And Preliminary Study Of Late Flowering Mutant Bdlf2 In Brachypodium Distachyon L.

Brachypodium distachyon L.belonging to the genus Bassica in the subfamily Brachypodium,has close genetic relationship with the gramineous plants,such as barley and wheat.It also has the characteristics of a series of model plants: small genomes,short growth cycles,simple growing conditions,and self-pollination.With the completion of the whole genome sequence of Bd21,the research on Brachypodium distachyon has been rapidly developed in recent years.The study using Brachypodium distachyon as model research materials of temperate gramineous plants have received more and more attention and favor.In order to speed up the research on functional genomics of Brachypodium distachyon,mutants are one of the most direct and effective ways to study gene function.Mutants are generated by physical mutagenesis,chemical mutagenesis(EMS mutagenesis),spontaneous mutations and insertional mutagenesis.At present,transposon tagging method is the most successful and effective method to obtain mutant materials.In order to conduct basic research on Brachypodium distachyon and identify key genes and sites controlling important agricultural traits accurately and effectively,we must first conduct forward genetics and reverse genetics on Brachypodium distachyon.The research on the positive and negative genetics of Brachypodium distachyon mutants will be the key to promote the development of gene function of Brachypodium distachyon.When a transposon is inserted into a functional gene region or a nearby site,the gene of the insertion site is inactivated or activated,and a mutant phenotype is induced.According to this characteristic,the relevant functional gene can be cloned using transposon tagging.Mutant screening in Brachypodium distachyon L.In order to obtain a large number of mutants of Brachypodium distachyon,to search for mutants related to flowering and agronomic traits in mutants,and to study the function and action mechanism of the mutant genes,we constructed an efficient Ac/Ds transposon tagging vector.The transposon tagging vector was transfected with Agrobacterium tumefaciens and the phenotypic observation and gene identification were performed on the offspring.At the same time,we screened the mutants of the progenies of the ethanol-inducible Ac/Ds transposition system transgenic seedlings.The main screening results are as follows:(1)Using the Ac/Ds transposition system to quickly obtain a large number of new insertion mutations,we have obtained several color mutants(yellowing,whitening,streaked spots),distortion(spikelet distortion,leaf distortion),dwarfing,developmental delay,disease,seed smaller,extreme late flowering and other mutants.Mutants were grown for at least 3 generations and the mutants were stable.Insertion element T-DNA or Ds was detected by PCR.Use Tail-PCR or reverse PCR to rapidly locate the T-DNA or Ds insertions.(2)Screening of progenies of transgenic seedlings with ethanol-induced Ac/Ds system has also resulted in some phenotypic mutants,such as albino seedlings,panicle distortion,dwarfing,etc.Preliminary study on mutant bdlf2The study of flowering time of plants has a crucial influence on the yield of the crop.We screened an extremely late mutant line,named bdlf2,and positioned T-DNA in the gap between the two genes by Tail-PCR.In order to find out the causes of late flowering phenotypes,we cloned and analyzed the genes on both sides.The main findings are as follows:(1)Observing the phenotype of the mutant bdlf2,it was found that it was extremely late flowering with strong vegetative growth ability,multi-branched,a growth cycle of more than one and a half years and a spikelet lengthening,the number of florets per spikelet increased and the florets were arranged closely.(2)qRT-PCR results showed that the expression levels of the left and right genes relative to the wild type were significantly upregulated.The left gene was found to be a hexokinase homologue gene,named BdHXK1.The amino acid sequence of the right gene contains an IQ67 conserved domain.The IQ67 domain contains a central motif of 67 conserved residues and is a hallmark feature of the plant specific IQD gene family.The right gene is named BdIQD1.(3)BdIQDI has the highest expression in the apical meristem and panicle.The expression of BdIQDI in leaves was relatively low,and the expression of BdIQDI was higher in the apical meristems of the four-leaf stage and panicles of the seven-leaf stage.(4)BdIQDI can affect spike length and fertility.Overexpression of BdIQDI phenotype resulted in longer spikelets,increased number of florets per spike,abnormal development of pistil structure,and a significant decrease in seed setting rate of transgenic plants.The structure of spikes overexpressing BdIQDI was similar to that of the mutant bdlf2,which confirmed that the change in the panicle structure of the mutant bdlf2 may be due to the upregulation of BdIQDI expression.The high expression level of BdIQDI combined with spikes suggested that BdIQDI plays an important role in the control of panicle development.(5)The key genes of flowering FT and VRN1 were significantly decreased in mutant bdlf2.The expression of FT and VRN1 of flowering promoting factor in bdlf2 was significantly decreased,but the expression of BdODDSOC1 and BdODDSOC2 of the flowering suppressor FLC in Arabidopsis was significantly upregulated.These indicate that changes in the expression levels of some flowering genes in the mutant bdlf2 may have caused late flowering.