טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentShamai Yeela
SubjectLeaf Senescence Promoters and Their Possible Use for the
Production of Recombinant Proteins in Plants
DepartmentDepartment of Biology
Supervisor Professor Emeritus Shimon Gepstein
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Leaf senescence constitutes the final stage of leaf development and is also characterized as nutrient relocation process from leaves to develop seeds. Leaf senescence involves a coordinated action at the cellular, tissue, organ, and organism levels under the control of a highly regulated genetic program. Understanding leaf senescence may also provide means to control leaf senescence for improving agricultural traits of crop plants and also for other non related subjects such as molecular farming. A wide spectrum of recombinant proteins is used in research, medicine and industry. Traditional production systems have drawbacks in terms of cost, scalability and product safety. Recent studies have shown that molecular farming in plants has many practical, economic and safety advantages compared to the conventional systems. One of the main challenges in molecular farming is the production of high yield functional recombinant proteins in plants. A major obstacle could be the toxicity of a foreign protein that might reduce the biomass or change normal plant development. To overcome this problem, we suggest employing suitable regulatory elements to direct the temporal pattern of protein synthesis. Our main objective was to design plant systems that efficiently produce recombinant proteins by improving plant biomass and synthesis of high yield foreign proteins only at late stages of the normal developmental. To achieve these goals, we used maturation - early senescence regulated promoters, ClpX, metallothionein and SARK, that direct the synthesis of the foreign proteins only after plant development is completed, and analyzed their temporal expression pattern in Arabidopsis and tobacco leaves by using the reporter gene GFP. Transgenic Arabidopsis plants containing the reporter gene under the control of ClpX and MT promoters showed the expected temporal expression, e.g. progressive expression with plant age. However, the SARK promoter drives only mRNA and not protein expression in the expected temporal pattern. GFP activity in tobacco plants under the control of ClpX promoter showed progressive expression with leaf development in contrast to constitutive activity with the MT promoter. Senescence delay and biomass improvement achieved by autoregulation of ckytokinin biosynthesis, by fusing the promoters to the gene isopentyl-transferase (IPT). Transgenic Arabidopsis, tobacco and tomato plants expressing the IPT gene under the control of the MT promoter showed remarkable biomass improvement accompanied by delayed senescence, without any morphological or developmental abnormalities. Our study suggests a suitable approach for the use of temporally regulated promoters for the development of efficient plant systems for the production of recombinant proteins.