Establishing A Highly Efficient System Of Shoot Regeneration Through Overexpressing Transcription Factor TaLAX1 In Wheat (Triticum Aestivum L.)

In vitro shoot regeneration is of crucial importance for genetic transformation.It has been shown that the frequency of shoot regeneration depends on genotypes,explants,culture conditions,e.g.in wheat(Triticum aestivum L.).At present,it’s critical to establish a highly efficient of shoot regeneration system mediated by some key regulators in wheat.In this study,a Ta LAX1 gene homologous with LAX PANICLE 1(LAX1)gene in rice was cloned and identified.Overexpression of Ta LAX1 could promote shoot regeneration and thus improve frequency of positive seedlings in wheat.The acquisition of transgenic wheat germplasm with a high shoot regeneration efficiency laid a foundation for the transformation of other excellent genes.And it will make great progresses in promoting the application of transgenic technology in improving wheat quality.The main results are as follows:(1)Phylogenetic tree analysis and identification of wheat Ta LAX1 geneWe identified a Ta LAX1 gene with up-regulated expression through transcriptome analysis of RNA-Seq data.Homologous sequence alignment and phylogenetic tree analysis showed the closest relationship between this gene and LAX1.Ta LAX1 gene was cloned successfully using stem tip c DNA as template from Chinese Spring wheat.(2)Expression pattern of Ta LAX1 geneTa LAX1 protein was localized in the nucleus.And it was highly expressed during callus differentiation when immature embryos of Chinese Spring wheat were used as explants for shoot regeneration induction.In situ hybridization showed that Ta LAX1 was specifically expressed in the spikelet primordia of double-ridge stage.It suggested that Ta LAX1 may play a role in regulating shoot regeneration and spikelet development of wheat.(3)Function of Ta LAX1 regulating shoot regeneration of wheatOverexpression of Ta LAX1-A gene could promote callus differentiation and increased the frequency of positive seedlings when transformed the p Ubi::Ta LAX1-A construct into immature embryos of Fielder,and it shortened the time of shoot emergence.Same effect was found when Ta LAX1-A overexpression construct was transformed into immature embryos of varieties with low regeneration efficiency of Shannong28 and Chinese Spring.It showed that the T1 generation transgenic plants of these two varieties of Fielder and Shannong28 with Ta LAX1-A overexpression construct had the powerful capacity to regenerate.These results suggested that Ta LAX1 had the function of regulating shoot regeneration.(4)Phenotypic analysis of transgenic plants with Ta LAX1-A overexpression constructIn addition,overexpression of Ta LAX1-A caused other phenotypes,such as about 15 days’ earlier heading time and fewer tillers.Taken together,Ta LAX1-A gene could regulate the heading time and the number of tillers,which was important for agricultural industry.