Cloning and characterization of TaVIP2 gene from Triticum aestivum and functional analysis in Nicotiana tabacum.

Lipu Du,Zhishan Lin,Pei Zhao,Xingguo Ye,Yunlong Zhang,Ke Wang,Wei Zhang

doi:10.1038/srep37602

Abstract

Wheat is recalcitrant to genetic transformation. A potential solution is to manipulate the expression of some host proteins involved in T-DNA integration process. VirE2 interacting protein 2 (VIP2) plays an important role in T-DNA transport and integration. In this study, a TaVIP2 gene was cloned from common wheat. Southern blot and allele-specific polymerase chain reaction (AS-PCR) combined with an online chromosomal location software tool revealed that three TaVIP2 genes were located on wheat chromosomes 1AL, 1BL, and 1DL. These three homoeoallelic TaVIP2 genes all contained 13 exons and 12 introns, and their coding sequences were the same; there were a few single nucleotide polymorphisms (SNPs) among the three genes. The heterologous expression of the TaVIP2 gene in tobacco led to enhancement of the Agrobacterium-mediated transformation efficiency up to 2.5-fold. Transgenic tobacco plants expressing TaVIP2 showed enhanced resistance to powdery mildew. Further quantitative real-time PCR (qRT-PCR) revealed that overexpression of TaVIP2 in transgenic tobacco up-regulated the expression of an endogenous gene, NtPR-1, which likely contributed to powdery mildew resistance in transgenic tobacco. Our study indicates that the TaVIP2 gene may be highly useful in efforts to improve Agrobacterium-mediated transformation efficiency and to enhance powdery mildew resistance in wheat.

Highlights

IntroductionOur study indicates that the TaVIP2 gene may be highly useful in efforts to improve Agrobacterium-mediated transformation efficiency and to enhance powdery mildew resistance in wheat
VIP1 is composed of 261 amino acids and contains a leucine zipper domain (a plant basic-zipper protein) that is homologous to most plant basic leucine zipper (bZIP) proteins containing a nuclear localization signal (NLS) in the conserved region of the leucine zipper domain[4]
These results demonstrated that VIP1 plays a vital role during the nuclear import of T-DNA in the Agrobacterium-mediated transformation process

Summary

Introduction

Our study indicates that the TaVIP2 gene may be highly useful in efforts to improve Agrobacterium-mediated transformation efficiency and to enhance powdery mildew resistance in wheat. Overexpression of AtVIP1 in transgenic tobacco plants dramatically enhanced T-DNA transformation efficiency when retransformed with Agrobacterium[6] These results demonstrated that VIP1 plays a vital role during the nuclear import of T-DNA in the Agrobacterium-mediated transformation process. When the VIP2-silenced transgenic tobacco and Arabidopsis plants were retransformed by Agrobacterium or biolistic particles, the stable transformation efficiencies declined, but their transient transformation efficiencies by the pathogen and stable transformation efficiencies by bombardment were not affected[7] This finding suggested that VIP2 is involved in the integration of T-DNA into the plant genome. Our work expands the knowledge and genetic resources and information available for wheat improvement

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Results

Conclusion