Construction of Vectors to Suppress Photorespiratory Pathway in Wheat and Reviews of Recombinant Protein Expression Technologies in Plants

摘要

Life on earth is sustained by a process of Photosynthesis.Plants utilise visible light spectra to produce biomass.Our environment is constantly changing which requires the photosynthetic machinery to work in more vigorous way to adopt itself to upcoming challenges of severe biotic and abiotic nature.To cope with these challenges, plant scientists are constantly working on the ways to enhance the photosynthetic activity.Photorespiration which fixes O2 and releases CO2 in C3 plants is the competing pathway with photosynthesis.One of the method to increase yield is to enhance photosynthesis through engineering photorespiratory pathway.A number of efforts have been carried out in this pursuit resulting in three engineered photorespiratory pathways to date, out of which two have been proven experimentally in model plant, Arabidopsis.These approaches may prove useful for field crops in future environment of more hostile nature.To experimentally prove this in wheat, six vectors are constructed expressing AtGDH, GCL and TSR aiming to install a novel pathway in wheat to supress photorespiration by gene gun.These vetors are PXJC-Ubi-TaCTP-GDH,PXJC-Ubi-HvCTP-GCL, PXJC-Ubi-ZmCTP-TSR, PXJC-Cmps-TaCTP-GDH, PXJC-Cmps-HvCTP-GCL, PXJC-Cmps-ZmCTP-TSR.These vectors are mainly divided into two groups based on Promoter.One group contain Ubiquitin promoter which is a strong promoter and shows very good results in wheat.The other one is with Cmps promoter.Three genes namely AtGDH, GCL, and TSR involved in novel pathway are codon optimised to wheat and synthesized synthetically.Additionally to make these genes express in Chloroplast different Chloroplast transit peptides from wheat barley and maize are used.These were then cloned in PXJC-Ubi-Upmi and PXJC-Cmps-Upmi vectors (Molecular Biotechnology Laboratory for Triticeae Crops).The plasmid as a whole or Clean Cassete can be used in gene gun transformation of wheat.A review is made on recent developments in protein expression technologies in plants,keeping in view the importance ofbio pharming as an emerging field.The infectious diseases along with cancers are the world leading causes of deaths in living organisms including human and animals.The last two decades have witnessed an incorporated endeavour in colligated biological sciences to cope with the problem in novel ways.With the advent of recombinant DNA technology in the early 1970's provided the grounds to produce recombinant proteins that can be used as therapeutic agents.A number of expression systems developed in the past to produce products of pharmaceutical value including bacterial and mammalian systems.Recently, advances are made in using plants as bioreactors to produce therapeutic proteins like vaccine antigens, antibodies including monoclonal antibodies,idiotype specific antibodies against infectious and cancerous diseases of high importance.This review will cover the recent advances in therapeutic protein expression in plants both stable (nuclear and chloroplast) as well as transient in comparisson to bacterial and mammalian cells, the factors affecting the heterologous protein expression, the vector systems and recent developments in existing technologies and the steps taken towards industrial production of plant made vaccines, antibodies and biopharmaceuticals.In continuation to the importance of bio pharming to developing countries in providing a cost effective fight against infectious and cancerous diseases another review is made titled Need of cost-effective vaccines in developing countries: what plants can offer?To treat present day's infectious diseases different therapies are used that include drugs or vaccines or combination of both.Currently, the world is facing an increasing problem of drug resistances from many pathogenic microorganisms.In the majority of cases, when vaccines are used, formulations consist of live attenuated microorganisms.This poses an additional risk of infection in immunocompromised patients and people suffering from malnutrition in developing countries.Therefore, there is a need to improve drug therapy as well as to develop next generation vaccines, in particular against infectious diseases with highest mortality rate.For patients in developing countries, costs related to treatments are one of the major hurdles to reduce the disease burden.In the present review, we describe some infectious diseases with heavy impact on health status of people in low and middle income countries.We discuss the prospects of plants as alternative platforms for the development of next-generation subunit vaccines that can be a cost-effective source for mass immunization of people in developing countries.This thesis is divided into three Chapters, The first chapter narrates the construction of vectors to suppress photorespiratoy pathway in plants, while Chapter 2 and 3 provides the reviews on Recent developments in protein expression technologies in plants and Need of cost-effective vaccines in developing countries: what plants can offer?

目录

声明

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

ABSTRACT

ABREVIATIONS

CHAPTERⅠ CONSTRUCTION OF VECTORS TO SUPPRESS PHOTORESPIRATORY PATHWAY IN WHEAT

1．1．Introduction

1．2 Photorespiration

1．2．1．The innate C3 photorespiratory pathway

1．2．2．Manipulation of Photorespiratory Pathway

1．3． Materials and Methods

1．3．1．Strains and Vectors

1．3．2．Culture Media for Bacteria

1．3．3．Preparation of liquid LB media 500 ml

1．3．4．Inoculation of media with bacteria

1．3．5．Plasmid DNA Isolation

1．3．6．Agarose Gel Electrophoresis

1．3．7．DNA Fragment Isolation from Agarose Gel

1．3．8．Restriction enzyme digestion and ligation reactions

1．3．9．Transformation of Bacteria

1．3．10．DNA Sequencing

1．4．Results

1．4．1．Restriction and ligation reactions

1．4．2．Vectors Constructions

1．5．Gene Gun Transformation in Wheat

1．5．1 Collection of Spikes；Grains Isolation And Sterelization

1．5．2 Sterelization of Grains In Clean Bench

1．5．3 Isolation Of Immature Embryo

1．5．4 Bombardment of Callus with AtGDH GCL and TSR fragments

1．6．Discussion

1．6．1．Rubisco and Photorespiration

References

CHAPTERⅡ RECENT DEVELOPMENTS IN PROTEIN EXPRESSION TECHNOLOGIES IN PLANTS

Abstract

Introduction

Background

Plants vs Other Heterologous Protein Expression Systems

Plants as Heterologous Protein Expression Systems to Fight Infectious and Cancerous Diseases

Strategies to Enhance Recombinant Protein Expression in Plants Transiently

Key Events in Development of Plant Derived Biopharmaceutics Industry

Key events in development of Plants derived Bio pharmaceutics

Conclusion

REFERENCES

CHAPTER Ⅲ NEED OF COST-EFFECTIVE VACCINES IN DEVELOPING COUNTRIES：WHAT PLANTS CAN OFFER?

Abstract

Introduction

Malaria

Tuberculosis

Hepatitis

Dengue

Pneumonia

Liver and cervical cancer

Need of Next Generation Subunit Vaccines

Plants as Cost-Effective Platforms for Vaccine Production

Conclusion

REFERENCES

Acknowledgments