Isolation and characterisation of genes involved in carbon and chlorophyll metabolism in Saccharum species hybrids

Fernhout, Jean-Jacque (2015-04)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: Sugarcane is a tropical perennial grass species belonging to the Poaceae (true grasses) family. Mature sugarcane is comprised mostly of sugarcane stalks, which accumulate high amounts of sucrose, a fact that has led to its wide cultivation of sugarcane for sucrose production. Sugar yields from sugarcane have been improved in the past by either creating transgenic sugarcane or through using traditional breeding methods. Increasing sugar yields in sugarcane is still of interest and new cisgenic strategies are being considered to alleviate consumer concerns over transgenic plants. This thesis consists of two parts. The first was aimed at understanding the relation between trehalose-6-phosphate (T6P) synthesis and sucrose accumulation in sugarcane. In this study the E. coli genes involved in trehalose synthesis, otsA and otsB, were overexpressed in sugarcane in order to observe their effects on soluble sugar accumulation. Nine otsA and two otsB overexpressing lines were created, confirmed by gDNA insertion PCRs, sq-RT-PCR and immuno detection of encoded enzymes. Preliminary measurements of soluble sugars showed that four out of the nine otsA lines had significantly decreased and one line significantly increased sucrose concentrations. Correlating sq-RT-PCR results with soluble sugar measurements suggest that trehalose-6-phosphate synthase (TPS) expression affects sucrose levels in sugarcane, but further research of TPS activity is required before a conclusion can be reached. Further analysis of mature cane material in regard to relevant enzyme levels, carbohydrate levels and gene expression should contribute to more conclusive results. Three novel sugarcane TPS encoding sequences were isolated and proven to be functional through complementation of the growth defect in tps1Δ yeast grown on glucose as a carbon source. Sugarcane TPS isoforms named SoTPSa, SoTPSb and SoTPSc, were isolated by successful application of 5‟ RACE alongside standard PCR using primers based on other monocotyledonous TPS sequences. The encoded SoTPSa contains a 25 amino acid insertion within the partial TPP domain. The encoded SoTPSc contains a 126 amino acid long N terminal truncation, which removes one of the thirteen amino acids found within the active site of the TPS domain. Future characterization of the encoded enzymes will determine the effects of these modifications on TPS activity. The second part of this thesis describes initial efforts made in attempting to develop a cisgenic in vitro selectable marker system for sugarcane, S. officinarum callus, which uses a diphenylether type (DPE) herbicide as a selection agent and a sugarcane protoporphyrinogen oxidase (PPO) gene as a selection marker. Firstly the plastid targeted PPO from tobacco (NtPPO-1) was isolated and mutagenized, to mimic the double mutated Arabidopsis PPO, used by Li et al., (2003) in maize. However, sugarcane calli transformed with the double mutated NtPPO-1 and grown on media containing fomesafen herbicide, were incapable of regenerating. Future efforts will utilize a N-terminal sequence that is targeted to the plastid organelle, so as to ensure translocation of the enzyme to that subcellular location. Also, random mutations were induced in the NtPPO-1 gene to screen for mutations that confer DPE herbicide resistance, however this work is currently on hold until a heme deficient E. coli can be obtained. Secondly, attempts were made to isolate a putative sugarcane plastid targeted PPO gene, so as to eventually use this in developing a cisgenic strategy. 5‟ RACE was successful in revealing additional nucleotide sequence adding 1006 bp to the already known partial sugarcane PPO sequence. However the fragment isolated was still a partial sequence.

AFRIKAANSE OPSOMMING: Suikerriet is 'n tropiese meerjarige gras spesie wat deel is van die Poaceae (ware grasse) familie. Volwasse suikerriet bestaan hoofsaaklik uit suikerrietstamme, wat hoë hoeveelhede sukrose akkumuleer, 'n feit wat gelei het tot die wye verbouing van suikerriet vir sukrose produksie. In die verlede is suikeropbrengste vanuit suikerriet verbeter deur die skep van transgeniese suikerriet óf die gebruik van tradisionele teelmetodes. Toenemende suiker opbrengste in suikerriet is steeds van belang en nuwe cisgeniese strategieë word oorweeg om verbruikerskommer oor transgeniese plante te akkommodeer. Hierdie tesis bestaan uit twee dele. Die eerste deel is daarop gemik om die begrip van die verhouding tussen trehalose-6-fosfaat (T6P) sintese en sukrose ophoping in suikerriet te verstaan. In hierdie studie is die E. coli gene wat betrokke is in trehalose sintese, otsA en otsB, ooruitgedruk in suikerriet ten einde die uitwerking daarvan in die opgaar van oplosbare suiker te bestudeer. Nege otsA en twee otsB verhoogte uitdrukkings lyne is geskep, bevestig deur gDNA bygevoegde PKR, sq-RT-PKR en immuno opsporing van geïnkripteerde ensieme. Voorlopige metings van oplosbare suikers toon dat vier van die nege otsA lyne ʼn beduidende afname in sukrose vlakke en een lyn „n beduidende toegeneem in sukrose vlakke getoon het. Korrelerende sq-RT-PKR resultate met oplosbare suikermetings dui daarop dat trehalose-6- fosfaat sintese (TPS) geenuitdrukking sukrose vlakke sal affekteer, maar verdere navorsing van TPS aktiwiteit is nodig voordat 'n gevolgtrekking gemaak kan word. Verdere ontleding van volwasse riet materiaal met betrekking tot relevante ensiem vlakke, koolhidrate vlakke en geenuitdrukking, behoort by te dra tot meer volledige resultate. In hierdie studie is drie nuwe suikerriet TPS gene geïsoleer en dit is bewys as funksioneel deur die komplimentering van die groeidefek van tps1Δ gis, gegroei op glukose as 'n koolstof bron. Suikerriet TPS isoforme, genoem SoTPSa, SoTPSb en SoTPSc, is geïsoleer deur die suksesvolle toepassing van 5 'RACE, in kombinasie met standaard PKR, deur van spesiaal ontwerpte primers, gebaseer op ander eensaadlobbige TPS gene, gebruik te maak. Die gekodeerde SoTPSa bevat 'n 25 aminosuur invoeging binne-in die gedeeltelike TPP domein. Die gekodeerde SoTPSc bevat 'n 126 aminosuur lange N terminaal afkapping, wat een van die dertien aminosure binne die aktiewe terrein van die TPS domein verwyder. Toekomstige karakterisering van hierdie geïnkripteerde ensiemes sal die effek van hierdie veranderinge op TPS aktiwiteit bepaal. Die tweede deel van hierdie tesis beskryf die aanvanklike probeerslae wat gemaak is in 'n poging om „n cisgeniese in vitro selekteerbare merker vir suikerriet, S. officinarum kallus te ontwikkel. Hierin word gebruik gemaak van 'n difenylether tipe (DPE) onkruiddoder as 'n seleksie agent, en 'n suikerriet protoporphyrinogen oksidase (PPO) geen as 'n seleksie merker. In 'n poging om dit te bewerkstellig is daar eerstens plastied geteikende PPO van tabak (NtPPO-1) geïsoleer en geteikende mutagenese suksesvol daarop uitgevoer. Mutasies wat geinduseer is, is gegrond op die dubbele gemuteerde Arabidopsis PPO, wat gebruik was in mielies deur Li et al., (2003). Alhoewel die suikerriet kallus getransformeer is met die dubbele gemuteerde NtPPO-1 konstruk en geselekteer is op media wat fomesafen onkruiddoder bevat, was die kallus nie in staat om te regenereer nie. In toekomstige pogings sal probeer word om 'n N-terminale volgorde, geteiken op „n plastied organel, te benut sodat translokasie van die ensiem aan die plastied organel verseker kan word. So ook is toevallige mutasies veroorsaak in die NtPPO-1 gene om te soek vir nuwe mutasies wat DPE onkruiddoderweerstand verleen, maar hierdie werk is tans gestop totdat 'n heem gebrekkige E. coli mutant verkry kan word. Tweedens, is pogings aangewend om 'n vermeende suikerriet plastied geteikende PPO gene te isoleer, om uiteindelik te gebruik in die ontwikkeling van 'n cisgeniese strategie in suikeriet. 5 'RACE was suksesvol in die onthulling van bykomende nukleotiede volgorde deur 1006 bp by te voeg by die reeds bekende gedeeltelike suikerriet PPO fragment. Nie teenstaande is die fragment wat nuut geïsoleer is, steeds slegs 'n gedeeltelike volgorde volgens vergelykings met ander bekende plant PPO gene.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/96696
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