Overexpression of a tomato SlNAC2 gene in Nicotiana tabacum to determine its potential role in enhancing drought tolerance in plants

Van Beek, Coenrad Roelof (2018-12)

Thesis (MSc)--Stellenbosch University, 2018.

Masters

ENGLISH ABSTRACT: The ability of transcription factors to regulate stress-response gene expression within a plant makes them ideal targets as transgenes for improving stress tolerance. The NAC (NAM/ATAF/CUC) transcription factor family has been well documented in their role in improving plant abiotic stress tolerance. Limited water resources for use in crop production present a significant threat to sustainable agriculture. The aim of this study was to overexpress the SlNAC2 (KT740994.1, Solanum lycopersicum NAC2) transcription factor in transgenic tobacco (Nicotiana tabacum). SlNAC2 was cloned into a modified pCambia1300 vector. Tobacco plants were transformed using Agrobacterium tumefaciens containing the pCambia1300:SlNAC2 vector. The first generation (T1) of transgenic plants were regenerated in vitro and selected using hygromycin. After selection plants were grown to maturity in the greenhouse, seeds were collected and putative transgenic lines analyzed for transgene insertion and expression. T1 seeds of three transgenic plants (T 4, 5 and 10) were geminated in vitro on selection and planted in the greenhouse for further analysis. A normal phenotype was seen when the plants were grown to maturity under watered conditions. When exposed to water stress for 21 days and then re-watered, all plants, displayed a stunted phenotype compared to non-stressed plants. At the flowering stage the control plants were significantly shorter than the transgenic plants. Control plants were also unable to form seed from every flower that they produced, whereas the transgenic plants could. Overexpression of SlNAC2 improved the transgenic tobacco plant’s ability to survive and recover from drought stress. Proline, chlorophyll and relative water content were found to be significantly higher in transgenic plant lines compared to the control plants. Oxidative stress and lipid peroxidation resulted in similar increased levels of H2O2, O2- and malonaldehyde in all plants. The dehydrated plants showed no significant difference in superoxide dismutase, catalase or reduced glutathione (GSH) activity or content between the transgenic and control plants. SlNAC2 overexpression did not seem to have a significant impact on stomatal conductance or chlorophyll fluorescence. sqPCR analysis indicated some increase in transcription of certain drought response marker genes at different stages within the drought stress period when compared to relative gene expression levels in the control plants. In the future, the potential relationship between SlNAC2 and abscisic acid and the systems they co-regulate, should be investigated in the transgenic plants overexpressing SlNAC2.

AFRIKAANSE OPSOMMING: Die vermoë van transkripsiefaktore om stres-respons-gene-uitdrukking binne 'n plant te reguleer, maak hulle ideale teikens as transgene om stresverdraagsaamheid te verbeter. Die NAC (NAM / ATAF / CUC) transkripsiefaktorfamilie is goed gedokumenteer in hul rol in die verbetering van plantabiotiese stresverdraagsaamheid. Beperkde waterhulpbronne vir gebruik in gewasproduksie bied 'n beduidende bedreiging vir volhoubare landbou. Die doel van hierdie studie was om die SlNAC2 (KT740994.1, Solanum lycopersicum NAC2) transkripsiefaktor in transgeniese tabak (Nicotiana tabacum) oor uit te druk. SlNAC2 is gekloneer in 'n aangepaste pCambia1300-vektor. Tabakplante is getransformeer met behulp van Agrobacterium tumefaciens wat die pCambia1300:SlNAC2-vektor bevat. Die eerste generasie (T1) transgeniese plante is in vitro geregenereer en gekies deur gebruik te maak van hygromisien. Nadat seleksieplante gegroei het tot volwassenheid in die kweekhuis, is sade versamel en veronderstelende transgeniese lyne ontleed vir transgeeninvoeging en uitdrukking. T1 sade van drie transgeniese plante (T4, 5 en 10) is in vitro ontkiem en gekies in die kweekhuis vir verdere analise. 'n Normale fenotipe is gesien toe die plante onder normale omstandighede tot volwassenheid gegroei het. Wanneer dit vir 21 dae aan waterstres blootgestel word en dan weer natgemaak word, het alle plante 'n verkorte fenotipe getoon in vergeleke met nie-gestresde plante. By die blomstadium was die beheerplante aansienlik korter as die transgeniese plante. Beheerplante was ook nie in staat om saad te vorm van elke blom wat hulle geproduseer het nie, terwyl die transgeniese plante kon. Ooruitdrukking van SlNAC2 het die transgeniese tabakplant se vermoë om te oorleef en te herstel van droogtestres verbeter. Proline, chlorofil en relatiewe waterinhoud was aansienlik hoër in transgeniese plantlyne in vergelyking met die beheerplante. Oksidatiewe stres en lipiedperoksidasie het gelei tot soortgelyke verhoogde vlakke van H2O2, O2- en malonaldehied in alle plante. Die gedehidreerde plante het geen beduidende verskil in superoksied dismutase, katalase of glutathion (GSH) aktiwiteit of inhoud tussen die transgeniese en kontrole plante gehad nie. SlNAC2 ooruitdrukking het nie 'n beduidende impak op stomatale geleiding of chlorofil-fluoresensie gehad nie. sqPCR analise het gedui op 'n mate van toename in transkripsie van sekere droogte-respons merker gene op verskillende stadiums binne die droogtestres periode wanneer dit vergelyk word met relatiewe geenuitdrukkings vlakke in die beheerplante. In die toekoms moet die moontlike verwantskap tussen SlNAC2 en absisiensuur en die stelsels wat hulle medereguleer, ondersoek word in die transgeniese plante wat SlNAC2 ooruitdruk.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105014
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