Production of libraries to study biopolymer metabolism in Arabidopsis thaliana and Tylosema esculentum

Swart, Corne (2013-12)

Thesis (MSc)--Stellenbosch University, 2013.

ENGLISH ABSTRACT: Biopolymers and bio-degradable polymers are of utmost importance to ensure a sustainable economy. Industry depends on raw material, which in many cases are derived from fossil fuels, but in light of looming energy crises and green revolutions attention is being directed at cellulose and starch biopolymers. This study was therefore set forth to investigate novel genetic key elements of cell wall metabolism in Arabidopsis thaliana and starch synthesis in an under-utilized southern African crop plant, Tylosema esculentum. In the first section of the study a cDNA library of good quality was constructed from regenerating A. thaliana protoplasts as it was expected to be enriching for genes involved in cell wall biosynthesis. Small scale EST sequencing of the library confirmed that a few sequences were similar to genes identified to be highly expressed during protoplast regeneration. The library was to be screened by expression in a microalgae as it is anticipated that cell wall metabolising genes would change the wall structure and visibly alter the colony morphology. An attempt was made at establishing a high-throughput transformation system in the unicellular algae Chlorella protothecoides in which the library was proposed to be screened. Conventional microalgal transformation techniques do not appear to be effective in this strain as the study produced no transgenic algae. Alternative studies into a screening system within another species could still lead to the identification of cell wall biosynthetic genes, which was the first objective in the study. The second objective in the study was to investigate the potential of the orphan crop T. esculentum as starch-producing cash-crop in developing southern African countries. In this section of the study a cDNA library of good quality was produced form the tuber of T. esculentum. The library was transferred to an expression vector and screened functionally in E. coli for the presence of sequences with starch synthase activity. No sequences have been identified yet and screening procedures are still on-going. The starch content in the tuber has also been determined for the first time. The relatively high starch content in combination with low agricultural inputs indicate the potential of the plant as an industrial starch source. Further investigations into the nature of the starch are proposed to identify prospective buyers within the industry.

AFRIKAANSE OPSOMMING: Biopolimere en bio-afbreekbare polimere is van kardinale belang om ‘n volhoubare ekonomie te ontwikkel. Industriële toepassings maak op die oomblik hoofsaaklik staat op fossielbrandstof verwante bronne, maar met die oog op ‘n groen revolusie en energie krissise wat dreig word meer belangstelling getoon in sellulose en stysel biopolimere. Hierdie studie is daarom onderneem om genetiese elemente te identifiseer wat betrokke is by die sintese van die selwand in Arabidopsis thaliana en stysel sintese in die suider Afrikaanse gewas Tylosema esculentum wat grotendeels onderbenut is. In die eerste deel van die studie is ‘n cDNA biblioteek, van goeie kwaliteit, geskep vanuit A. thaliana protoplaste wat besig was om hulle selwande te herbou. Dit word verwag dat die protoplaste gedurende die tydperk aktief besig sal wees om gene uit te druk wat betrokke is by selwandsintese. DNA volgordebepaling het bevestig dat ‘n klein aantal volgordes ooreengestem het met gene wat voorheen gevind was om in ‘n oormaat uitgedruk te word tydens die herbou van protoplas-selwande. Daar was beoog om die biblioteek in ‘n mikroalge uit te druk en sodoende die morfologie op kolonievlak waar te neem vir verandering wat in die selwand meegebring is. Om hierdie rede was die doel om ‘n hoë opbrengs transformasie sisteem te ontwikkel in die mikroalge Chlorella protothecoides. Algemene mikroalge transformasie tegnieke blyk om nie effektief in die spesie te wees nie aangesien geen transgeniese alge waargeneem is nie. Die ontwikkeling van ‘n soortgelyke proses in ‘n ander spesie kan steeds lei na die ontdekking van gene betrokke by selwandsintese in A. thaliana wat die eerste uitkoms van die projek as geheel was. Die tweede uitkoms van die projek was om te ondersoek wat die waarskynlikheid was om T. esculentum te kommersialiseer as ‘n stysel gewas en sodoende ‘n inkomste te skep vir arm boere in ontwikkelende lande in suider Afrika. In hierdie gedeelte van die projek was daar ‘n goeie cDNA biblioteek geskep uit die knol van T. esculentum. Die biblioteek is oorgedra na ‘n plasmied waarop dit aktief uitgedruk kon word in Escherischia coli G6MD2 en daar is gesoek na volgordes wat lei na die sintese van stysel in hierdie bakterieë. Tot op hede is geen sulke volgordes gevind nie, maar die ondersoek gaan steeds voort. Die styselinhoud van die knol is ook vir die eerste keer bepaal in hierdie ondersoek. ‘n Styselinhoud wat relatief hoog is en die lae moeite wat geverg word om die gewas te verbou toon dat die plant potensieel het as ‘n kommersiële bron van stysel. Verdere ondersoeke in die aard van die stysel word ook voorgestel om toekomstige industriële kopers te identifiseer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/85607
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