ABSTRACT
In recent years, gene engineering is developing rapidly and many recombinant proteins have been expressed. The use of plant bioreactor to express specific pharmaceutical proteins provides a new way for the prevention and treatment of some important diseases in human beings. Nowadays, chloroplast genetic transformation and expression system has become a research hotspot in plant bioreactor. Higher plant chloroplasts have unique advantages in the expression of recombinant proteins due to their special structures and inherited characteristics: such as high expression, site-specific integration, and the maternal inheritance characteristics of exogenous genes. The maternal inheritance of chloroplast is helpful for biological safety of transgene escaping by pollens. Many important pharmaceutical proteins have been successfully expressed in plant chloroplasts. As a chloroplast transformation model of higher plants, tobacco has made significant progress in the expression of pharmaceutical proteins, such as vaccine antigens, antibodies, and other important recombinant proteins. Chloroplast genetic transformation in higher plants also provides new techniques and methods for the study of chloroplast gene expression and regulation mechanisms. In order to provide a new idea for the development of chloroplast expression platform and the expression of important pharmaceutical proteins, this review outlined the progress of chloroplast genetic transformation system in higher plants, including the chloroplast transformation principle, vector construction, expression of recombinant proteins and important pharmaceutical proteins, and the effects of recombinant proteins expression on plant metabolism and traits.
ABSTRACT
Background: Transgenic plants inhabiting single Bt gene are prone to develop insect resistance and this resistance has been reported in case of some important yield-devastating insect larvae of commercial crops, such as cotton and rice. Therefore, it has become essential to adapt new strategies to overcome the problem of insect resistance and these new strategies should be sophisticated enough to target such resistant larvae in broad spectrum. Among these, plants may be transformed with Bt gene tagged with some fusion-protein gene that possesses lectin-binding capability to boost the binding sites for crystal protein gene within insect mid-gut in order to overcome any chances of insect tolerance against Bt toxin. Enhanced chloroplast-targeted Bt gene expression can also help in the reduction of insect resistance. Results: In the present investigation, a combined effect of both these strategies was successfully used in cotton (G. hirsutum). For this purpose, plant expression vector pKian-1 was created, after a series of cloning steps, carrying Cry1Ac gene ligated with chloroplast transit peptide towards N-terminal and Ricin B-Chain towards C-terminal, generating TP-Cry1Ac-RB construct. Conclusions: Efficacy of pKian-1 plasmid vector was confirmed by in-planta Agrobacterium-mediated leaf GUS assay in tobacco. Cotton (G. hirsutum) local variety MNH-786 was transformed with pKian-1 and the stable integration of TP-Cry1Ac-RB construct in putative transgenic plants was confirmed by PCR; while fusion-protein expression in cytosol as well as chloroplast was substantiated by Western blot analysis. Whereas, confocal microscopy of leaf-sections of transgenic plants exposed that hybrid-Bt protein was expressing inside chloroplasts.