Identification and manipulation of starch synthases from marama bean and potato

Hartzenberg, Lionel (2016-12)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Starch is a major storage carbohydrate in plants and algae. It consist of amylose and amylopectin and provides energy during heterotrophic growth. The accumulation of starch differs between certain families. Viridiplantae (plants and green algae) utilize ADP-glucose for starch synthesis and store it within the chloroplast, whereas Rhodophyceae (red algae) utilize UDP-glucose and both synthesize and store starch in the cytoplasm. Starch is a commercial product of worldwide importance and research into improving current starch sources and examining alternatives may provide benefits to the starch industry. This study consisted of two parts. The first involved increasing the physical and genetic information available for the southern African legume Tylosema esculentum. This involved obtaining and analysing transcriptomic data in order to find genes relating to starch synthesis. The second part aimed to randomly mutagenize a Solanum tuberosum SSI gene for the utilization of UDP-glucose instead of ADP-glucose for starch synthesis. Tylosema esculentum plants were grown for approximately one year before the leaf and tuber material was harvested. RNA extraction and cDNA synthesis was performed and the samples were sent for next-generation sequencing at the Agricultural Research Council (Pretoria). Reads were compiled and trimmed to produce three contigs related to starch metabolism. Using the Phytozome soybean database, we selected three genes (granule-bound starch synthase (GBSS), starch synthase 2 (SSII), starch synthase 4 (SS4)), and soybean-like actin, relating to starch synthesis within soybean. We matched them to open reading frames within the marama bean transcriptome and designed primers for their amplification from cDNA. All were successfully amplified. We also obtained additional information on the physical properties of T. esculentum starch granules. Two methods were performed in order to accurately obtain data on the marama bean starch granule size distribution. Both showed that the starch granule size is predominantly between 15-35 μm, with no particles above 50 μm. A reducing end assay was also performed to determine the amylose and amylopectin average chain length of marama bean starch. The amylose chain was found to be 67.5% longer than that of harvested potato starch, and 31.8% longer than that of commercial grade potato starch (Sigma-Aldrich). The amylopectin chain length was found to be 34.8% lower than that of harvested potato starch, but similar to that of commercial grade potato starch. The higher accumulation of long amylose chains could account for previous Rapid Visco Analyser (RVA) analyses performed that showed marama bean starch having unique viscosity properties, and may in turn lead to several other novel uses for marama bean. In the second part of this study, we amplified the UGPase (UDP-glucose pyrophosphorylase) gene from Arabidopsis thaliana to facilitate the production of UDP-glucose. The UGPase gene was ligated into the pACYC 184 vector and transformed into G6MD2, an E. coli strain lacking the glg operon necessary for glycogen metabolism. The S. tuberosum SSI gene within pBluescript.SK was randomly mutagenized using XL1-red E. coli cells, which are deficient in 3 of the primary proof reading mechanisms. Of these mutagenized plasmids, 33 were pooled and analysed against the unaltered SSI gene as template. We obtained 192 mutations in total, 4 per kb, with 98 point mutations, 57 insertions and 37 deletions. The SSI library was transformed into G6MD2::pACUG and screened for activity via iodine vapour. 150 000 colonies were screened but no expressing colonies were found. In the future alternative mutation methods as well as larger colony screenings may yield better results.

AFRKAANS OPSOMMING: Stysel is 'n groot stoor-koolhidraat in plante en alge. Dit bestaan uit amilose en amilopektien en verskaf energie tydens heterotrofiese groei. Die opeenhoping van stysel verskil tussen sekere families. Viridiplantae (plante en groen alge) gebruik ADP-glukose vir stysel sintese en stoor dit in die chloroplast, terwyl Rhodophyceae (rooi alge) UDP-glukose gebruik en albei sintetiseer en stoor stysel in die sitoplasma. Stysel is 'n kommersiële produk met wêreldwye belang en navorsing in die verbetering van die huidige styselbronne en die ondersoek van alternatiewes kan voordele inhou vir die styselbedryf. Hierdie studie het bestaan uit twee dele. Die eerste deel omvat die toename in die fisiese en genetiese inligting wat beskikbaar is vir die Suid-Afrikaanse peulplant Tylosema esculentum. Dit behels die verkryging en ontleding van transkriptomiese data om gene wat verband hou met stysel sintese op te spoor. Die tweede deel was gemik om „n Solanum tuberosum SSI gene lukraak te muteer vir die benutting van UDP-glukose in plaas van ADP-glukose vir stysel sintese. Tylosema esculentum plante was gekweek vir ongeveer een jaar voordat die blaar en knol materiaal geoes. RNS ekstraksie en cDNA sintese was uitgevoer en die monsters is gestuur vir volgende-generasie volgorde-bepaling by die Landbounavorsingsraad (Pretoria). Reekse is saamgestel en afgewerk om drie samestellings te produseer wat verwant is aan stysel metabolisme. Deur gebruik te maak van die Phytozome sojaboon databasis, het ons drie gene gekies (granule-gebinde stysel sintase (GBSS), stysel sintase 2 (SSII), stysel sintase 4 (SS4)), asook sojaboon-agtige aktien, verwant aan stysel sintese binne sojabone. Ons het hulle ooreengestem met oop leesrame binne die marama boontjie transkriptoom en inleiers is ontwerp om hulle te amplifiseer vanaf kDNS. Almal was suksesvol geamplifiseer. Ons het ook bykomende inligting verkry oor die fisiese eienskappe van T. esculentum styselgranules. Twee metodes is uitgevoer om die data op die marama boontjie styselgranule grootte-verspreiding akkuraat te bekom. Beide het getoon dat die styselgranule-grootte is hoofsaaklik tussen 15-35 μm, met geen deeltjies bo 50μm. „n Reduserende endtoets is ook uitgevoer om die amilose en amilopektien gemiddelde kettinglengte van marama boontjie stysel te bepaal. Die amilose ketting is gevind om 67,5% langer as dié van geoeste aartappelstysel en 31,8% langer as dié van kommersiële graad aartappelstysel (Sigma-Aldrich) te wees. Die amilopektien kettinglengte is gevind om 34,8% laer as dié van geoeste aartappelstysel te wees, maar soortgelyk aan dié van kommersiële graad aartappelstysel. Die hoër opeenhoping van lang amilosekettings kan van rekenskap gee vir die vorige RVA ontledings wat uitgevoer was en aangedui het dat marama boontjie stysel unieke viskositeit-eienskappe het, en kan op sy beurt lei tot verskeie ander nuwe gebruike vir die marama boontjie. In die tweede deel van hierdie studie, het ons die UGPase (UDP-glukose pyrophosphorylase) geen van Arabidopsis thaliana geamplifiseer om die produksie van UDP-glukose te fasiliteer. Die UGPase geen is afgesnoer in die pACYC 184 vektor en omskep in G6MD2, 'n E. coli stam sonder die glg operon wat nodig is vir glikogeen metabolisme. Die S. tuberosum SSI gene binne pBluescript.SK is lukraak muteer met behulp van XL1-rooi E. coli selle, wat gebrekkig is in 3 van die primêre proeflees meganismes. Van hierdie muteerde plasmiede, is 33 saamgevoeg en teen die onveranderde SSI gene as sjabloon ontleed. Ons het 192 mutasies in totaal verkry, 4 per kb, met 98 puntmutasies , 57 invoegings en 37 skrappings. Die SSI biblioteek is omskep in G6MD2::pACUG en gesif vir aktiwiteit via jodium dampe. 150 000 kolonies is ondersoek, maar geen uitdrukking kolonies is gevind nie. In die toekoms kan alternatiewe metodes van mutasie asook groter kolonie siftings beter resultate oplewer.

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