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

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 <i>in vivo</i>, we usually need tubulin and MAP co-expressed. Here, we constructed a dual-transgene vector expressing rice (<i>Oryza sativa</i>) 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 <i>GFP</i> fragment, <i>α-tubulin</i> open reading frame (ORF), and <i>NOS</i> 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 <i>CaMV 35S</i> promoter, <i>mCherry</i> fragment, and <i>NOS</i> 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, <i>MAP</i> ORF can be cloned into the site of 5' or 3' terminus of <i>mCherry</i> 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 <i>MAP</i> genes including <i>GL7</i>, <i>OsKCBP</i>, <i>OsCLASP</i>, and <i>OsMOR1</i> were cloned into the vector respectively, transformed into <i>Agrobacterium tumefaciens</i> strain, and expressed in <i>Nicotiana benthamiana</i> 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.