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A Pipeline for Exogenous Gene Expression and Biomass Analysis in Plants Bijay Bisunke Eastern Illinois University

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A Pipeline for Exogenous Gene Expression and Biomass Analysis in Plants Bijay Bisunke Eastern Illinois University Eastern Illinois University The Keep Masters Theses Student Theses & Publications 2019 A Pipeline for Exogenous Gene Expression and Biomass Analysis in Plants Bijay Bisunke Eastern Illinois University Recommended Citation Bisunke, Bijay, "A Pipeline for Exogenous Gene Expression and Biomass Analysis in Plants" (2019). Masters Theses. 4417. https://thekeep.eiu.edu/theses/4417 This Dissertation/Thesis is brought to you for free and open access by the Student Theses & Publications at The Keep. It has been accepted for inclusion in Masters Theses by an authorized administrator of The Keep. For more information, please contact [email protected]. The Graduate School~ EA< n K.'i h 111-.01\ UN11'f..RS11 r Thesis Maintenance and Reproduction Certificate FOR : Graduate Candidates Completing Theses in Partial Fulfillment of the Degree Graduate Faculty Advisors Directing the Theses RE : Preservation, Reproduction, and Distribution of Thesis Research Preserving, reproducing, and distributing thesis research is an important part of Booth Library's responsibility to provide access to scholarship. In order to further this goal, Booth Library makes all graduate theses completed as part of a degree program at Eastern Illinois University available for personal study, research, and other not-for­ profit educational purposes. Under 17 U.S.C. § 108, the library may reproduce and distribute a copy without infringing on copyright; however, professional courtesy dictates that permission be requested from the author before doing so. 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A PIPELINE FOR EXOGENOUS GENE EXPRESSION AND BIOMASS ANALYSIS IN PLANTS By Bijay Bisunke THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOLOGICAL SCIENCES IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY CHARLESTON, ILLINOIS 2019 I HEREBY RECOMMEND THAT THIS THESIS BE ACCEPTED AS FULFILLING THIS PART OF THE GRADUATE DEGREE CITED ABOVE ~ R DATE DEPAR;ENT CHAIR DA TE 't-ltr.!t? THESJSCOMMITTEEMEMBER ~ATE THESIS COMMITTEE MEMBER DATE Abstract Biological pretreatment of biomass is typically required for the production of liquid biofuels, which are viable alternatives to traditional fossil fuels. While traditional thermomechanical pretreatments are effective at helping to deconstruct lignocellulose, they are often expensive and tend to generate environmentally dangerous waste materials. An alternative to traditional pretreatment strategies is biological pretreatment, which has focused on lignocellulose-degrading organisms, such as white-rot fungi. While direct pretreatment of biomass with white-rot fungi has shown great promise, directly expressing functional white-rot fungi genes in plants (i.e. in plan/a expression) has the potential to be even more efficient because the biomass itself would produce the deconstruction enzymes, which would decrease the amount of steps toward biofuel production. This thesis details an efficient pipeline for cloning and expressing genes in planta followed by protocols for assessing changes to carbohydrates and lignin chemistry. An In-Fusion cloning system was developed for Agrobacterium-based transformation into Arabidopsis thaliana using green fluorescent protein as a representative gene. Modified acid hydrolysis and thioacidolysis procedures were used to explore cellulose and lignin chemistry on pretreated (i.e. non-traditional) biomass. Overall, this research generated a procedural work-flow for the rapid cloning and transformation of exogenous genes into Arabidopsis, followed by reliable carbohydrate analysis of the resulting biomass. This pipeline is expected to be used in future studies examining the expression of enzymes involved in biomass deconstruction from white-rot fungi into commercially-relevant plant biomass. TABLE OF CONTENTS 1. Introduction .. .... .......................... .... ........................... .... ... .... ... ........ ........................... ...... 1 2. Research Objectives .... ... .. ...... ..... ...... .. ............. ............ ... ... ....... ......... ................ ............. 5 3. Materials and Methods ............................................................. ................ ....................... 6 3 .1. Agrobacterium-based Transformation of Arabidopsis thaliana ............ .................... .. 6 3 .1.1 . Creation of Competent Agrobacterium cells .. ...... ... .. .... .................... .... ... ................. 6 3.1.2. Transformation of competent cells ......................... .... ...... .................... ........... .... ... .. 6 3 .1.3 . Plasmid Isolation and Transformation Validation .... ... ... ... .............. ...... ........ 7 3.1.4. Transformation of Arabidopsis thaliana wi th Agrobacterium tumefaciens .. 8 3.1.5. Seed Germination and Selection ......... ..................... .. ... .. .. ..... ........................ 9 3 .2. Pretreatment and Analysis of Biomass ... ....... .. .. ..... ... ............. .. ....................... ............. 9 3.2.1. Biomass Sources ................ .... .. ........... .. .... .. .............. .. ........ ..... .... .... ... ........... 9 3 .2.2. Fungal Growth and Biomass Inoculation ... ... ....................................... ...... 10 3.2.3 . Preparation of the Biomass for Chemical Analysis ..................................... 10 3.2.4. Acid Hydrolysis ............ ... ......... .... .................. ...... .................. ............ .. .. .. ... 11 3.2.5. Thioacidolysis ..................... ... .................... ... ... ............. ... ...... .................... .. 11 4. Results and Discussion ............... ... ....... .......................................................... .... ..... ... ... 12 4.1. Transformation Efficiency ...... ........................... .... ......................................... 12 4.2 Lignocellulose Analysis ....... ....... ................... ........................................................... ... 13 5. Concl us ion .. .. ............................... .. .. ...... .. .. .. .. .... ... ... .. ... .... ... ... ......... ......... ..................... 15 6. References .......... .. ..... ... ... .. .. ... ... ............. .................... ... ... .. ............. .. ................. ........ .... 23 LIST OF FIGURES Figure 1. Detailed structure of lignocellulose ......................... .............. ............................... 2 Figure 2. General outline of the research pipeline ................................ .. ............................. 6 Figure 3. Map of pGFP ... ..................................................................................................... 7 Figure 4. Chemical structures of guaiacyl and syringyl lignin .. ... ... .................................. 12 Figure 5. Total number of seeds sprouted in Arabidopsis thaliana ................................... 16 Figure 6. Second generation of transformed A. thaliana ................................................... 17 Figure 7. Glucose content after acid hydrolysis of biomass ................... ........................... 18 Figure 8. Xylose content after acid hydrolysis of biomass ................. .... ... ........................ 19 Figure 9. Galactose content after acid hydrolysis of biomass ............ ............................... 20 Figure 10. Arabinose content after acid hydrolysis of biomass ......................................... 21 Figure 11. S/G ratio after thioacidolysis .........................................
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