Functional Analysis and Marker Development of TaCRT-D Gene in Common Wheat (Triticum aestivum L.).

Jiping Wang,Ruilian Jing,Runzhi Li,Xinguo Mao

doi:10.3389/fpls.2017.01557

Abstract

Calreticulin (CRT), an endoplasmic reticulum (ER)-localized Ca2+-binding/buffering protein, is highly conserved and extensively expressed in animal and plant cells. To understand the function of CRTs in wheat (Triticum aestivum L.), particularly their roles in stress tolerance, we cloned the full-length genomic sequence of the TaCRT-D isoform from D genome of common hexaploid wheat, and characterized its function by transgenic Arabidopsis system. TaCRT-D exhibited different expression patterns in wheat seedling under different abiotic stresses. Transgenic Arabidopsis plants overexpressing ORF of TaCRT-D displayed more tolerance to drought, cold, salt, mannitol, and other abiotic stresses at both seed germination and seedling stages, compared with the wild-type controls. Furthermore, DNA polymorphism analysis and gene mapping were employed to develop the functional markers of this gene for marker-assistant selection in wheat breeding program. One SNP, S440 (T→C) was detected at the TaCRT-D locus by genotyping a wheat recombinant inbred line (RIL) population (114 lines) developed from Opata 85 × W7984. The TaCRT-D was then fine mapped between markers Xgwm645 and Xgwm664 on chromosome 3DL, corresponding to genetic distances of 3.5 and 4.4 cM, respectively, using the RIL population and Chinese Spring nulli-tetrasomic lines. Finally, the genome-specific and allele-specific markers were developed for the TaCRT-D gene. These findings indicate that TaCRT-D function importantly in plant stress responses, providing a gene target for genetic engineering to increase plant stress tolerance and the functional markers of TaCRT-D for marker-assistant selection in wheat breeding.

Highlights

Calreticulin (CRT) is an abundant Ca2+-binding protein predominantly localized in the endoplasmic reticulum (ER)
The results showed that there was no difference in germination rate between transgenic lines and wild-type seeds. 76.8% wild-type seed germinated after 12 days in MS medium supplemented with 75 mM NaCl, while 95.7% seeds of TaCRT-Doverexpression transgenic Arabidopsis line &1 germinated, 97.1% seeds of line &2 germinated, and 88.4 and 46.4% seeds of lines &3 and &4 germinated, respectively
To investigate further whether TaCRT-D is involved in other stress responses, we developed a number of transgenic Arabidopsis lines overexpressing the ORF of TaCRT-D

Summary

Introduction

Calreticulin (CRT) is an abundant Ca2+-binding protein predominantly localized in the endoplasmic reticulum (ER). A typical CRT protein contains three distinct structural and functional domains: a highly conserved globular N-domain which includes a cleavable signal peptide sequence; a P-domain which is responsible for high affinity and low capacity Ca2+-binding ability; and a C-domain which includes a (K/H)DEL. ER retrieval signal (Michalak et al, 1999, 2009) and exhibits low affinity and high capacity Ca2+-binding ability. Extensive studies of CRTs have elucidated many key physiological functions inside and outside the ER (Michalak et al, 1999, 2009; Gold et al, 2010; Wang J.P. et al, 2012; Wang W.A. et al, 2012), such as intracellular Ca2+ storage, the regulation of ER Ca2+ homeostasis (Mery et al, 1996; Mesaeli et al, 1999; Persson et al, 2001), involvement in Ca2+-dependent signal pathways (Nakamura et al, 2001; Arnaudeau et al, 2002; Gelebart et al, 2005), molecular chaperone activity in the ER (Nigam et al, 1994; Nauseef et al, 1995; Jin et al, 2009; Jiang et al, 2014), control of cell adhesion (Opas et al, 1996; Johnson et al, 2001), angiogenesis (Ding et al, 2014), functions related to the immune system and apoptosis (Waterhouse and Pinkoski, 2007) as well as roles in pathogenesis (Qiu and Michalak, 2009)

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Discussion

Conclusion