Construction and validation of co-expression vector for rice alpha tubulin and microtubule associated protein respectively fused with fluorescent proteins

Abstract

Microtubule (MT) consists of α-tubulin and β-tubulin. The dynamic instability regulated by various microtubule associated proteins (MAPs) is essential for MT functions. To analyze the interaction between tubulin/MT and MAP in vivo, we usually need tubulin and MAP co-expressed. Here, we constructed a dual-transgene vector expressing rice (Oryza sativa) α-tubulin and MAP simultaneously. To construct this vector, plant expression vector pCambia1301 was used as the plasmid backbone and Gibson assembly cloning technology was used. We first fused and cloned the GFP fragment, α-tubulin open reading frame (ORF), and NOS terminator into the vector pCambia1301 to construct the p35S::GFP-α-tubulin vector that expressed GFP-α-tubulin fusion protein. Subsequently, we fused and cloned the CaMV 35S promoter, mCherry fragment, and NOS terminator into the p35S::GFP-α-tubulin vector to generate the universal dual-transgene expression vector (p35S::GFP-α-tubulin-p35S::mCherry vector). With the p35S::GFP-α-tubulin-p35S::mCherry vector, MAP ORF can be cloned into the site of 5' or 3' terminus of mCherry to co-express GFP-α-tubulin and MAP-mCherry/mCherry-MAP. To validate the availability and universality of the dual-transgene expression vector, a series of putative rice MAP genes including GL7, OsKCBP, OsCLASP, and OsMOR1 were cloned into the vector respectively, transformed into Agrobacterium tumefaciens strain, and expressed in Nicotiana benthamiana leaves. The results indicated that all of the MAPs were co-expressed with α-tubulin and localized to MTs, validating the availability and universality of the vector and that GL7, OsKCBP, OsCLASP, and OsMOR1 might be MAPs. The application of the co-expression vector constructed by us would facilitate studies on the interaction between tubulin/MT and MAP in tobacco transient expression systems or transgenic rice.

Keywords: Co-expression vector; Fluorescent protein; Microtubule; Microtubule associated protein; Rice; Tubulin.

Figure 1. Construction of p35S::GFP-α-tubulin vector and its expression in Nicotiana benthamiana leaf epidermal cells.

(A) Schematic of pCambia1301 vector. (B) Schematic of p35S::GFP-α-tubulin vector. (C) Subcellular localization of GFP protein. (D) Subcellular localization of GFP-α-tubulin as the p35S::GFP-α-tubulin vector is expressed. All micrographs are projections of confocal Z-stacks and bar = 10 μm.

Figure 2. Construction of p35S::GFP-α-tubulin-p35S::mCherry vector and its expression in Nicotiana benthamiana leaf epidermal cells.

(A) Schematic of p35S::GFP-α-tubulin-p35S::mCherry vector. (B) Subcellular localization of GFP-α-tubulin as the vector is expressed. (C) Subcellular localization of mCherry as the vector is expressed. (D) Micrograph of GFP-α-tubulin merged with mCherry. All micrographs are projections of confocal Z-stacks and bar = 10 μm.

Figure 3. Co-expressions of rice α-tubulin and MAPs as the dual-transgene vectors are transformed into Nicotiana benthamiana leaf epidermal cells respectively.

(A) Co-expressions of rice GFP-α-tubulin and mCherry-GL7. (B) Co-expressions of rice GFP-α-tubulin and mCherry-OsKCBP. (C) Co-expressions of rice GFP-α-tubulin and mCherry-OsCLASP. (D) Co-expressions of rice GFP-α-tubulin and OsMOR1-mCherry. All micrographs are projections of confocal Z-stacks and bar = 10 μm.