Cloning And Function Analysis Of Several Genes Relevant To Agrobacterium-mediated Transformation And Tissue Culture Regeneration In Wheat

Successful and stable transformation procedure is an essential step for the transgenic breeding of wheat. Because of the low efficiencies of plant regeneration and genetic transformation, transgenic research in wheat fall behind other crops. Among the several developed transformation techniques, Agrobacterium-mediated way is the most efficient technique. However, Agrobacterium-mediated transformation is really a complicated biological process, in which a lot of Agrobacterium protein and plant proteins interact with each other for the mission of importing, transportation and integration of T-DNA. Related host proteins play important roles almost in each step of the transformation. Several associated genes with transformation and plant regeneration are planed to be cloned and identified using molecular biology methods in this study, which will significantly influence the genetic engineering and cell engineering breeding of wheat.VIP2 is a plant protein that was reported to assist T-DNA transformation in tobacco. Using At VIP2 as query, Ta VIP2 was isolated from common wheat by in silico strategy. The c DNA for Ta VIP2 is 1839 bp in length and encodes 612 amino acids which have 48% similarity with the correspondence protein of At VIP2 in Arabidopsis. Southern blotting analysis revealed that three copies of Ta VIP2 were harbored in common wheat which were located on the chromosome of 1A, 1B and 1D using a set of substitution lines between durum wheat and Ae. tauschii. Sequences of c DNA and g DNA of the three Ta VIP2 genes were achieved from three diploid wild species of wheat and the allelic Ta VIP2 genes all contained 13 exons and 12 introns. Subcellular localization analysis in onion epidermal cells indicated that the Ta VIP2 protein were localized to the plasma membranes, cytoplasm and nucleus. Ta VIP2 was specifically interacted with Vir E2 from Agrobacterium using yeast two-hybrid technology. By heterologous expression in tobacco, Ta VIP2 gene was identified to improve the Agrobacterium-mediated transformation efficiency of tobacco. Especially, the transgenic tobacco plants expressing TaVIP2 showed enhanced resistance to powdery mildew.VIP1 is an another reported plant protein that was reported to assist T-DNA transformation in tobacco. Using the same strategy, Ta VIP1 was isolated from common wheat. The c DNA for Ta VIP1 is 987 bp in size, and encodes 328 amino acids, which have 51% similarity with the correspondence protein of At VIP1 in Arabidopsis. Southern blotting analysis revealed that three copies of Ta VIP1 were harbored in wheat which were located on the chromosome of 5A, 5B and 5D based on the mapping ESTs in common wheat database. Subcellular localization analysis in onion epidermal cells indicated that the Ta VIP1 protein was localized to the plasma membranes, cytoplasm and nucleus. Heterologous expression results demonstrated that the transferring of Ta VIP1 gene has nothing to do with the Agrobacterium-mediated transformation efficiency of tobacco.WUS was a transcription factor regulating the formation of stem cells and related to plant regeneration in Arabidopsis. Sequences of c DNA and g DNA of Ta WOX5 gene were cloned from common wheat byPCR, which were 630 bp and 749 bp in length, respectively. Ta WOX5 gene contained 2 exons and 1 introns, and encodes 209 amino acids which have 39% similarity with the correspondence protein of At WUS in Arabidopsis. Southern blotting analysis revealed that more than 9 copies of Ta WOX5 were harbored in wheat. Ta WOX5 gene were transferred into tobacco and wheat using Agrobacterium-mediated transformation. Results suggested that overexpression of Ta WOX5 in wheat could increase the width of leaves and tiller number, and promotes the growth of nutritional organs in tobacco.Expression of histone was proved to enhance transformation efficiency in tobacco and rice. Ta Histone H1 and Ta Histone H2 B gene were targeted and isolated using RNA-Seq technology. These two genes were transferred into tobacco and wheat via Agrobacterium-mediated method. And the obtained transgenic plants will be further tested for function verification of Ta Histone H1 and Ta Histone H2 B genes.