Stable Expression of Basic Fibroblast Growth Factor in Chloroplasts of Tobacco.

Zheng-Yi Wei,Yu-Ying Zhang,Xiao-Fang Zhong,Chun-Jing Lin,Yan-Zhi Liu,Shao-Chen Xing,Yu-Hong Cai,Jian Ma,Yun-Peng Wang

doi:10.3390/ijms17010019

Abstract

Basic fibroblast growth factor (bFGF) is a multifunctional factor in acceleration of cell proliferation, differentiation and transference, and therefore widely used in clinical applications. In this study, expression vector pWX-Nt03 harboring a codon-optimized bFGF gene was constructed and introduced into the tobacco chloroplasts by particle bombardment. After four rounds of selection, bFGF was proved to integrate into the chloroplast genome of regenerated plants and two of four transgenic plants were confirmed to be homoplastomic by PCR and Southern hybridization. ELISA assay indicated that bFGF represented approximately 0.1% of total soluble protein in the leaves of transplastomic tobacco plants. This is the first report of bFGF expression via chloroplast transformation in model plant, providing an additional option for the production of chloroplast-produced therapeutic proteins.

Highlights

Fibroblast growth factors (FGF) are a large family of heparin-binding protein mitogens, with potential therapeutic utilities due to their broad target cells specificity
No plastid-produced products have been used in clinical trials yet, vaccines produced by nuclear transformation of plants or TMV-based transient expression in plants have been tested in the clinic [23,29]
BFGF has previously been expressed by nuclear transformation system and the yield, 2.3% TSP in soybean seeds and 9.55% TSP in rice seeds [15,16], is rather higher than the production in the present experiment, but considering that: (1) the biomass of tobacco mature leaves with average yield of 2.7 tons per hectare [31]; (2) the tobacco needs approximately 40–50 days from seed to fully-expanded leaves; and (3) it is practicable to harvest five to seven times annually under field condition depending on the growing season, the Basic fibroblast growth factor (bFGF) productivity of transgenic tobacco might be more competitive compared with soybean and rice

Summary

Introduction

Fibroblast growth factors (FGF) are a large family of heparin-binding protein mitogens, with potential therapeutic utilities due to their broad target cells specificity. One FGF, basic fibroblast growth factor (bFGF or FGF2), a single-subunit protein with a molecular weight of ~17 kDa and a pI of 9.6, was first cloned from the bovine brain and pituitary in 1978 [2]. Recombinant bFGF is highly desirable in the marketing due to its critical role in the growth of blood vessels, wound healing, tissue regeneration and nerve system [6,7,8] and many attempts have been made to meet the great demand over the past few decades. Two main crops, soybean and rice, were reported to express functional recombinant bFGF in seeds. IInn tthhiiss ssttuuddyy,, wwee eennggiinneeeerreedd ttoobbaaccccoo ppllaannttss ttoo eexxpprreessss bbFFGGFF vviiaa ppllaassttiidd ttrraannssffoorrmmaattiioonn. TToo oouurr kknnoowwlleeddggee,, ththisisisitshethfiersftirrsetporertpoofrbt FoGf FbeFxGpFresesxepdrevsisaepdlavstiiad ptrlaanstsifdortmraantisofonramnadtitohnereafnodre tphreorveifdoeres aprnoovviedleaslatenrnoavteivl aelotefrbnFaGtivFeeoxpf breFsGsiFone.xpression

RReessuullttss

Discussion

Plant Materials

Vector Construction for Chloroplast Transformation

Chloroplast Transformation and Homoplastomic Selection

Molecular Testing

Findings

Western Blot Assay