Establishment Of Plant Regeneration System And Creation Of Large Grain Germplasms Using TaLEC Genes In Wheat(Triticum Aestivum L.)

Wheat(Triticum aestivum L.)is one of the three major food crops in the world.Agrobacterium-mediated genetic transformation is the main means of gene function research in wheat,but the low plant regeneration ability limits the establishment of efficient genetic transformation and hinders the effective development of gene function analysis and genetic engineering breeding in wheat.The establishment of efficient regeneration system is of great significance for functional genomics and molecular design breeding of wheat.In this study,we cloned the homologs of transcription factor LEAFY COTYLEDON 1(LEC1)and LEC2 from wheat,named TaLEC1 s and TaLEC2 s,and performed the phylogenetic tree and spatio-temporal expression pattern analyses.Finally,the biological function of TaLEC1 and TaLEC2 genes in wheat were investigated.The main results are as follows:The full-length amino acid sequences of of Arabidopsis LEC2 were used to search wheat sequence data in Ensembl Plants database using the BLASTP retrieval tool.It was found that TaLEC2 genes were located on chromosome 3,named TaLEC2-3A ? TaLEC2-3B and TaLEC2-3D,respectively.specific primers were designed and three TaLEC2 genes were obtained by cloning using RT-PCR technology using the embryos of wheat(Cultivar Chinese spring)cv.Chinese spring and Fielder as materials.Their deduced peptipes contain 248,405 and 301 amino acids residues,respectively.TaLEC2 s contain typical the B3 domain.Phylogenetic tree analysis indicated that TaLEC2 s and LEC2 were in the same branch.The subcellular localization result showed that TaLEC2 proteins were located in the nucleus.The result of qRT-PCR analysis revealed that TaLEC2 genes were expressed in the embryo,shoot apical meristem(SAM)at double-ridge stage and spikelet primordium of cv.Chinese spring,with highest level in the embryo.Further,in situ hybridization experiments showed that TaLEC2 genes were expressed specifically in the young embryo,seed coat as well as endosperm of cultivar Chinese spring.The sequence information of TaLEC1 was obtained through the same strategy as above.The result showed that the genes with the highest similarity to Arabidopsis LEC1 in cv.Chinese spring were located on 6A?6B and 6D chromosomes.Specific primers were designed and TaLEC1 genes were cloned by RT-PCR technology from young embryos of Chinese spring and Fielder and named TaLEC1-6A,TaLEC1-6B and TaLEC1-6D.All three TaLEC1 s encode 246 amino acids residues and contain the HAP3 domain.Phylogenetic tree analysis indicated that TaLEC1 s and rice OsLEC1 were in the same branch and shared high homology.Subcellular localization results showed that TaLEC1 proteins were localized in the nucleus.The result of qRT-PCR analysis showed that TaLEC1 genes were expressed in different degrees in the embryo,SAM at single-ridge stage and double-ridge stage of wheat cv.Chinese spring.In situ hybridization results showed that TaLEC1 genes were expressed specifically in the embryos of wheat.To investigate the biological function of TaLEC1 and TaLEC2 genes in wheat,TaLEC1 and TaLEC2 overexpression vector,amiRNA vector,estrogen-induced expression vector and TaLEC2 antisense RNA expression vector were constructed and Agrobacterium tumefaciens mediated genetic transformation was carried out with young embryos of cv.Fielder as receptor materials.So far,TaLEC2-3A-OX,TaLEC2-3D-OX and TaLEC2-3D-DOWN generation positive plants have been obtained,and T0 generation positive plants transformed by TaLEC2-3B-OX,amiRNATaLEC2 and estrogen-induced expression vectors have been obtained.qRT-PCR results showed that the expression level of TaLEC2-3A and TaLEC2-3D was up-regulated overexpression plants,and the expression level of TaLEC2 s was down-regulated in TaLEC2-3D-DOWN transgenic plants.Compared with the Fielder plant,TaLEC2-3A-OX and TaLEC2-3D-OX transgenic wheat plants showed slow growth,lower plant height and less tiller numbers.However,TaLEC2-3D-DOWN transgenic plants were robust,plant height and tillers increased.Interestingly,TaLEC2-3D-DOWN plants produced larger grains with length and width and grain weight increased obviously in comparison with Fielder plants,which indicated that reducing the expression level of TaLEC2 could effectively increase the grain size and yield per plant.Morphological and histological observations revealed that overexpression TaLEC2-3D induced somatic embryogenesis and embryogenic cell formation and maintainance.,and overexpression of TaLEC1 s promoted embryonic cellformation and maintainanceTaken together,TaLEC2 s overexpression in wheat induced the production of embryogenic cell and somatic embryogenesis in vitro,and TaLEC1 s overexpression promoted embryogenic cell formation and maintenance.Using antisense RNA technology to reduce the expression level of TaLEC2 genes could remarkably increase grain size and grain weight in wheat.Our results were useful for the establishment of efficient genetic transformation based on the efficient somatic embryo regeneration.More importantly,the result provided important ideas and clues for wheat high yield breeding and molecular design breeding by use of gene editing technology to artificially manipulate the expression level of TaLEC2 s in wheat.