Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: The aim of this study was toe stablish a reliable protocol for the production 0 f transgenic pearl millet as this will open new avenues for augmenting the gene pool of this crop. This was achieved by identifying a highly regenerabie genotype and optimisation of a tissue culture system, and biolistic protocol f or stable integration of selected transgenes. Both a negative, herbicide resistance selectable marker gene, bar, and a positive selectable marker gene, manA, were individually introduced in order to identify and establish a reliable transformation protocol. The optimised transformation protocol was then used to introduce an antifungal gene in the genome of pearl millet to enhance resistance to the biotrophic fungus Sclerospora graminicola. S. graminicola, an obligate oomycetous fungal phytopathogen, is the causal agent of downy mildew in pearl millet plants and a major constraint in the production of pearl millet. A single component of antifungal resistance was introduced into the genome of pearl millet, as preliminary work towards determining its role in the total plant defence system. The approach chosen was to introduce a hydrolytic enzyme, 13-1,3- glucanase, from Trichoderma atroviride (formerly T. harzianum), a soil-borne filamentous fungus, capable of parasitizing several plant pathogenic fungi. It was anticipated that introducing this glucanase gene from T. atroviride which degrades glucan in the fungal cell walls, would significantly contribute to the improvement of resistance against downy mildew. Constructs were prepared containing the gene (gluc78) encoding a 78 kDa beta-1,3- glucanase. The constructs were prepared containing the gluc78 gene driven either by a strong constitutive promoter (ubiquitin promoter, exon and intron) or a wound inducible promoter, the potato proteinase inhibitor ilK gene promoter. The wound inducible promoter includes either an AMV leader' sequence or the rice Act1 intron to obtain higher expression levels in the monocotyledonous plant. The transformation efficiency using the particle inflow gun and the herbicide resistance gene, bar, was improved from 0.02% on a MS based medium, to 0.19 or 0.72% with manA as selectable marker gene on MS or L3 based medium, respectively. However, individual experiments, introducing manA as selectable marker gene, resulted in frequencies of 1.2 and 3%. This translated to one transformation event per plate, which contains on average 31-35 pre-cultured immature zygotic embryos. This is the first report of t he successful introduction and expression of a 13-1,3-glucanase encoding gene from a biocontrol fungus not only under constitutive expression but also under wound inducible expression in a plant. Optimisation of genetic engineering of pearl millet, a cereal crop recalcitrant to transformation, and the introduction of an antifungal transgene, was accomplished in this study. Initial results hint that expression of this transgene enhances resistance to S. graminicola. / AFRIKAANSE OPSOMMING: Die doel van die studie was om 'n betroubare genetiese transformeringsprotokol vir pêrel manna te ontwikkel. Hiervoor moes eerstens 'n regenereerbare genotipe geidentifiseer word. Twedens moes 'n betroubare weefselkultuur en biolistiese transformeringssisteem ontwikkel word. Beide die onkruiddoder bestandheidsgeen, bar, en 'n positiewe selektiewe geen, manA, is vir die doel van die projek onafhanklik in die genoom van pêrel manna in gekloneer. Die optimale sisteem is vervolgens aangewend om 'n geen wat potensieël verbeterde bestandheid teen die biotrofiese swam Sclerospora graminicola wat donsige meeldou by plante veroorsaak, in pêrel manna in te kloneer. 'n Enkele komponent van bestandheid is in die genetiese material van pêrel manna in gekloneer as inleidende werk om die rol van hierdie geen in die totale verdedigingsisteem te bepaal. Die benadering wat gekies was, behels die klonering van 'n hidrolitiese ensiem 13-1,3-glukanase, van Trichoderma atroviride (voorheen T. harzianum), 'n grondgedraagde swam, wat op 'n aantal ander plantpatogene fungus kan parasiteer. Die verwagting is dat klonering van hierdie 13- 1,3-glukanase geen van T. atroviride wat die glukaan verteer in die selwande van swamme, 'n groot verbetering tot die bestandheid teen donsige meeldou sal meebring. Konstrukte is voorberei wat die gluc78 geen bevat wat kodeer vir die 78 kDa beta-1,3-glukanase protein. Die konstrukte wat voorberei is bevat die gluc78 geen geinduseer deur of 'n sterk konstituwe promoter (ubiquitin promoter, exon en intron) of deur 'n wond geinduseerde promoter, die aartappel proteinase inhibeerder ilK geen promoter. Hierdie promoter word gevolg deur of 'n AMV leier volgorde of die rys Act1 intron om verhoogde uitdruk vlakke in monokotiele plante te verseker. As die partikel invloei geweer in kombinasie met die onkruiddoderbestandheidsgeen gebruik word, was die doeltreffendheid van transformasie 0.02% op 'n MS gebasseerde groeimedium. 'n Transformasie doeltreffendheid van onderskeidelik 0.19 en 0.72% is verkry wanneer die manA as selektiewe geen gebruik is op MS of L3 gebasseerde medium. Twee individual eksperimente, waar die manA geen as selektiewe geen gebruik is, het gelei tot 'n transformasie doeltreffendheid van 1.2 of 3%. Dit gee 'n gemiddelde van een transformasie per plaat wat 31 tot 35 voorafgekweekte onvolwasse embrios bevat. Hierdie is d ie eerste verslag van d ie suksesvolle klonering en uitdrukking van 'n 13-1,3-glukanasekoderende geen van 'n swam wat as 'n biologiese beheeragent gebruik word. Hierdie is nie alleenlik onder konstitutiewe uitdrukking nie, maar ook 0 nder wond g einduseerde u itdruk in' n p lant. In hierdie studie is die 0 ptimisering van genetiese verbetering van pêrel manna, 'n graan gewas wat gehard is teen transformasie, deur die klonering van 'n bestandheidsgeen in die genoom van hierdie gewas gedoen. Aanvanklike resultate dui daarop dat die uitdruk van hierdie geen lei tot verbeterde bestandheid teen S. graminicola.

published_or_final_version / abstract / Botany / Doctoral / Doctor of Philosophy

Šio darbo tikslas buvo patikrinti ar įmanoma padidinti mielių Saccharomyces cerevisiae K2 tipo kilerinio preprotoksino geno raiškos galimybes augaluose, naudojant konstitutyvų žiedinio kopūsto mozaikos viruso CaMV 35S promotorių. Šiam tikslui pasiekti buvo atlikta: neonkogeninių agrobakterijų (Agrobacterium tumefaciens) kamieno konjugantų atrinkimas, turinčių augalų transformacijos vektorių pART27-KillK2 su mielių K2 tipo kilerinį preprotoksiną koduojančiu genu; atlikta paprastojo tabako (Nicotiana tabacum L.) transformacija minėtų agrobakterijų pagalba bei selektyvi Nicotiana tabacum L. transgeninio kaliaus atranka. Po sėkmingos Agrobacterium tumefaciens konjugacijos su E.coli kamienu, turinčiu kilerinę plazmidę pART27-KillK2 ir sėkmingos modelinio augalo N.tabacum agrobakterinės lapų eksplantų transformacijos, buvo taikomas PGR metodas bei vykdomas kileriškumo patikrinimas. PGR ir DNR elektroforezės pagalba buvo nustatyta, kad KillK2 genas yra įsiterpęs į augalo genomą ir vyksta silpna šio geno raiška, tačiau žiedinio kopūsto mozaikos viruso CaMV 35S promotoriaus prieš šį geną aptikti nepavyko. Ankstesnių tyrimų metu buvo manoma, kad K2 tipo kilerinio preprotoksino geno informacija transformuotuose augaluose su vektoriumi pGA482-KillK2 yra realizuojama kuomet šio geno transkripcija tiesiogiai reguliuojama mielių alkoholdehidrogenazės ADH1 promotoriumi, tačiau šio darbo metu, atliekant tyrimus, buvo parodyta, kad promotorius ADH1 taip pat nėra prisijungęs prie geno KillK2... [toliau žr. visą tekstą] / The aim of this work was to verify the possibility to increase expression of yeast (Saccharomyces cerevisiae), K2 type killer preprotoxin in plants, using constitutive cauliflower mosaic virus CaMV 35S promoter. In order to achieve it, the following was done: selection of unoncogenic strain of agrobacterium (Agrobacterium tumefaciens) with plant transformation vector pART27-KillK2, containing K2 type killer preprotoxin gene; transformation of plain tobacco (Nicotiana tabacum L.) with mentioned agrobacteria; selection of Nicotana tabacum L. transgenic callus. Following the success of the Agrobacterium tumefaciens conjugation with E.coli strain (containing killer plasmid pART27-KillK2) and successful transformation of model N.tabacum plant with A.tumefaciens, there was used the PCR method to check out the killer effect of it. PCR and DNA electrophoresis showed that KillK2 gene is incorporated into plant genome and there is weak expression of this gene. However there was not found the promoter of couliflower mosaic virus CaMV 35S upstream this gene. In the previous studies, it was considered that the expression of mentioned transformed gene exist only when the transcription of it is directly regulated by alcoholdehidrogenase ADH1 promoter. However after investigation it was shown that promoter ADH1 isn’t connected to beginning of gene KillK2. There is the possibility that both ADH1 and CaMV35S promoters are remote from the beginning of KillK2 gene, or the iniciation is done by... [to full text]

Due to the stochasticity, diversity and variability of gene expression, transgenic crop study, is confronted with some uncertainties, such as what kinds of the influence from foreign gene on the transgenic crop, and how to fulfill the monitoring of transgenic crop growth real-/ near real-time efficiently. The influence of foreign gene could be treated as a special source of stress to vegetation. Therefore, it is promising to detect the difference between transgenic and contrast group and so as to monitor the growth of sample to assist to fulfill sample screening work, focusing on the plant biophysical traits or responses to stress by spectral techniques. Hyperspectral remote sensing technique is a kind of practical and field spectroscopy technique, which is simple, rapid, real-/ near real-time, user friendly and cheap. In this study, this technique was employed to indicate the differences between transgenic crop samples and their parents, and to monitor their growth. By the proposed approach, fine spectra of transgenic paddy rice were obtained, and the growth of samples were monitored the by their biophysical traits, finally the screening of cultivars were fulfilled in contrast controlled experiments. The biophysical traits or bio-process were concentrated on rather than on micro-structure or components of proteins. It will be implemented to monitor the growth of the samples real-/ near real-time, helping researchers know their samples clearly and screen samples efficiently. / In order to develop and validate this approach, 6 experiments in different fields were conducted, including three kinds of genomes and their transgenic samples. They were classified as the experiment-repeat experiments and the gene-repeat experiments. Moreover, a three-month experiment was also conducted for evaluating the capability of the approach to monitor the sample growth under the condition of an artificial stress (herbicide stress). Morphologic and parameterized features of foliar spectra of samples were applied to indicate the growth of the samples. / In the future, more factors should be considered. They are mainly: much more effective communication with biological researchers should be conducted; more research methods should be introduced, the study scope should be extended to the whole bands (350-2500nm) and more foliar chemicals should be involved as indicators of the growth status of the samples, etc. ii / The results proved this approach proposed was not a substitute to the popular methods for gene detection and crop assessment, but an important, helpful and efficient complement to make the crop breeding study under control and efficient as much as possible. By the approach, the researcher could know their samples clearly and real-/near real- time. / Li, Ru. / Advisers: Jinsong Chen; Hui Lin. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

by Cheng Man Kin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 71-76). / Abstracts in English and Chinese. / Thesis committee --- p.i / Abstract --- p.ii / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of contents --- p.vii / List of figures --- p.x / List of tables --- p.xi / Chapter Chapter 1: --- General introduction --- p.1 / Chapter Chapter 2: --- Literature review --- p.3 / Chapter 2.1 --- Nutritional quality of plant proteins --- p.3 / Chapter 2.2 --- Using traditional plant breeding method to enhance amino acid quality of plant proteins --- p.3 / Chapter 2.3 --- Molecular strategies to enhance amino acid quality of plant proteins --- p.4 / Chapter 2.3.1 --- Heterologous gene expression --- p.5 / Chapter 2.3.2 --- Protein sequence modification --- p.8 / Chapter 2.3.3 --- Modification of biosynthesis pathway --- p.10 / Chapter 2.3.4 --- Synthetic gene expression --- p.11 / Chapter 2.3.5 --- Homologous gene overexpression --- p.13 / Chapter 2.4 --- Arabidopsis --- p.14 / Chapter 2.4.1 --- Arabidopsis as a model plant --- p.14 / Chapter 2.4.2 --- Transformation methods --- p.14 / Chapter 2.4.2.1 --- Direct DNA uptake --- p.15 / Chapter 2.4.2.2 --- Agrobacterium-mediated transformation --- p.15 / Chapter 2.5 --- Winged Bean Lysine-Rich protein --- p.17 / Chapter 2.5.1 --- Identification of winged bean polypeptides rich in lysine --- p.17 / Chapter 2.5.2 --- Cloning of the lysine-rich protein gene --- p.17 / Chapter 2.5.3 --- Further characterization of the WBLRP gene --- p.18 / Chapter 2.6 --- Phaseolin --- p.19 / Chapter Chapter 3: --- Expression of LRP in transgenic Arabidopsis --- p.20 / Chapter 3.1 --- Introduction --- p.20 / Chapter 3.2 --- Materials and methods --- p.21 / Chapter 3.2.1 --- Targeting LRP to cytosol --- p.21 / Chapter 3.2.1.1 --- Chemicals --- p.21 / Chapter 3.2.1.2 --- Plant materials --- p.21 / Chapter 3.2.1.3 --- Bacterial strains --- p.22 / Chapter 3.2.1.4 --- Construction of chimeric LRP gene (pBILRP-1) --- p.22 / Chapter 3.2.1.4.1 --- PCR amplification of LRP --- p.22 / Chapter 3.2.1.4.2 --- Cloning of PCR-amplified LRP into vector pD3-8 --- p.26 / Chapter 3.2.1.4.3 --- Cloning of recombinant plasmid pLRP-1 into binary vector --- p.26 / Chapter 3.2.1.5 --- Transformation of Agrobacterium with pBILRP-1 --- p.27 / Chapter 3.2.1.6 --- Vacuum infiltration transformation of Arabidopsis --- p.28 / Chapter 3.2.1.7 --- Selection of transgenic plants --- p.29 / Chapter 3.2.1.8 --- GUS assay --- p.30 / Chapter 3.2.1.9 --- DNA isolation --- p.31 / Chapter 3.2.1.10 --- PCR amplification and detection of transgenes --- p.31 / Chapter 3.2.1.11 --- Southern blot hybridization --- p.31 / Chapter 3.2.1.12 --- RNA isolation --- p.32 / Chapter 3.2.1.13 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.32 / Chapter 3.2.1.14 --- Protein extraction and SDS-PAGE --- p.33 / Chapter 3.2.1.15 --- Protein sequencing --- p.33 / Chapter 3.2.1.16 --- Amino acid analysis --- p.34 / Chapter 3.2.2 --- Targeting LRP to protein bodies --- p.35 / Chapter 3.2.2.1 --- Chemicals --- p.35 / Chapter 3.2.2.2 --- Plant materials --- p.35 / Chapter 3.2.2.3 --- Bacterial strains --- p.35 / Chapter 3.2.2.4 --- Construction of chimeric LRP gene (pBILRP-2) --- p.35 / Chapter 3.2.2.4.1 --- Site-directed mutagenesis --- p.36 / Chapter 3.2.2.4.2 --- Cloning of the mutated phaseolin fragment into pBluescript --- p.36 / Chapter 3.2.2.4.3 --- PCR amplification of LRP --- p.39 / Chapter 3.2.2.4.4 --- Insertion of LRP into plasmid pBK/phas* --- p.39 / Chapter 3.2.2.4.5 --- Insertion of plasmid pLRP-2 into Agrobacterium binary vector --- p.41 / Chapter 3.2.2.5 --- Transformation of Agrobacterium with pBILRP-2 --- p.41 / Chapter 3.2.2.6 --- Vacuum infiltration transformation of Arabidopsis --- p.41 / Chapter 3.2.2.7 --- Selection of transgenic plants --- p.41 / Chapter 3.3 --- Results and discussion --- p.42 / Chapter 3.3.1 --- Targeting LRP to protein bodies --- p.42 / Chapter 3.3.1.1 --- Morphology of transgenic Arabidopsis --- p.42 / Chapter 3.3.1.2 --- Selection of transgenic plants --- p.42 / Chapter 3.3.2 --- Targeting LRP to cytosol --- p.46 / Chapter 3.3.2.1 --- Morphology of transgenic Arabidopsis --- p.46 / Chapter 3.3.2.2 --- Selection of transgenic plants --- p.46 / Chapter 3.3.2.3 --- Detection of GUS activity --- p.49 / Chapter 3.3.2.4 --- Integration of LRP transgene into Arabidopsis genome --- p.54 / Chapter 3.3.2.5 --- LRP transcript in transgenic Arabidopsis --- p.58 / Chapter 3.3.2.6 --- Stable accumulation of LRP in transgenic Arabidopsis --- p.61 / Chapter 3.3.2.7 --- Amino acid analysis of seed protein --- p.64 / Chapter Chapter 4: --- General discussion --- p.67 / Conclusion --- p.70 / References --- p.71

Long, Xiaohang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 146-165). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Wong, Hon-Kit. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 119-139). / Abstract in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.vii / General abbreviations --- p.ix / Abbreviations of chemicals --- p.x / List of figures --- p.xii / Table of contents --- p.xvi / Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Nitrogen assimilation and regulation in plants --- p.1 / Chapter 1.2 --- Asparagine metabolism and its gene regulation in plants --- p.2 / Chapter 1.2.1 --- A brief introduction of asparagine --- p.2 / Chapter 1.2.2 --- Asparagine synthetase gene family in A. thaliana --- p.3 / Chapter 1.2.3 --- Reciprocal regulation of ASN1 and ASN2 gene --- p.3 / Chapter 1.2.4 --- Primary structure difference of ASN1 and ASN2 protein --- p.4 / Chapter 1.2.5 --- "ASN1 overexpressor support the notion that it is a major gene regulating the free asparagine levels in plant tissues, while ASN may play different physiological function(s)" --- p.2 / Chapter 1.2.6 --- Evidence support ammonium-dependent AS in plant --- p.6 / Chapter 1.3 --- Ammonium toxicity and mechanism of ammonium toxicity to plant --- p.7 / Chapter 1.3.1 --- Ammonium toxicity --- p.7 / Chapter 1.3.2 --- Mechanism of ammonium toxicity --- p.9 / Chapter 1.4 --- "Relationship among asparagine, ammonium, and stress physiology" --- p.12 / Chapter 1.4.1 --- Ammonium accumulates under stress conditions --- p.12 / Chapter 1.4.2 --- Asparagine accumulates under stress conditions --- p.14 / Chapter 1.5 --- Relationship of asparagine metabolism and photorespiration --- p.17 / Chapter 1.5.1 --- A brief introduction of photorespiratory pathway --- p.17 / Chapter 1.5.2 --- Involvement of Asn in the photorespiration nitrogen cycle --- p.18 / Chapter 1.5.3 --- Reassimilation of ammonium released from photorespiration --- p.19 / Chapter 1.5.4 --- Photorespiration and stress physiology --- p.21 / Chapter 1.6 --- Role of amino acids in abiotic stress resistance --- p.23 / Chapter 1.6.1 --- Overview --- p.23 / Chapter 1.6.2 --- Proline accumulation and plant adaptation to water deficits and salinity stress --- p.24 / Chapter 1.6.3 --- Role of amino acids as precursors of quaternary ammonium compounds serving as compatible osmolytes --- p.28 / Chapter 1.7 --- A brief history of protoplast transient expression systems --- p.35 / Chapter 1.8 --- Advantages of mesophyll protoplast transient expression systems --- p.37 / Chapter 1.9 --- Hypothesis and main idea of this study --- p.38 / Chapter 2 --- Methods and Materials --- p.39 / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Plants --- p.39 / Chapter 2.1.2 --- Bacterial strains and plasmid vector --- p.39 / Chapter 2.1.3 --- Primer used --- p.39 / Chapter 2.1.4 --- Chemicals and reagents used --- p.40 / Chapter 2.1.5 --- Solution used --- p.40 / Chapter 2.1.6 --- Commercial kits used --- p.40 / Chapter 2.1.7 --- Equipment and facilities used --- p.40 / Chapter 2.2 --- Methods --- p.41 / Chapter 2.2.1 --- Growth medium and condition --- p.41 / Chapter 2.2.1.1 --- Normal growth condition --- p.41 / Chapter 2.2.1.2 --- Growth medium and stresses treatments --- p.41 / Chapter 2.2.1.3 --- Plant growth in Azaserine medium --- p.43 / Chapter 2.2.2 --- Biochemical Assay --- p.44 / Chapter 2.2.2.1 --- Ammonium assay --- p.44 / Chapter 2.2.2.2 --- Ammonium extraction for ammonium assay --- p.46 / Chapter 2.2.2.3 --- Soluble protein determination --- p.46 / Chapter 2.2.2.4 --- Detection of chlorophyll content --- p.47 / Chapter 2.2.3 --- Molecular techniques --- p.47 / Chapter 2.2.3.1 --- Bacterial cultures for recombinant DNA --- p.47 / Chapter 2.2.3.2 --- Recombinant DNA techniques --- p.48 / Chapter 2.2.3.3 --- Transformation of DH5a Competent cell --- p.48 / Chapter 2.2.3.4 --- Gel electrophoresis --- p.49 / Chapter 2.2.3.5 --- DNA and RNA extraction from plant tissues --- p.50 / Chapter 2.2.3.6 --- Generation of cRNA probes for Northern blot analyses --- p.52 / Chapter 2.2.3.7 --- Northern blot analysis --- p.53 / Chapter 2.2.3.8 --- PCR techniques --- p.54 / Chapter 2.2.3.9 --- Sequencing --- p.55 / Chapter 2.2.4 --- Genetic techniques --- p.56 / Chapter 2.2.4.1 --- Backcross of Azaserine resistant mutant --- p.56 / Chapter 2.2.4.2 --- Phenotype screening of backcross progenies --- p.56 / Chapter 2.2.5 --- Transient gene expression --- p.57 / Chapter 2.2.5.1 --- Protoplast isolation from Arabidopsis leave --- p.57 / Chapter 2.2.5.2 --- Protoplast transformation --- p.58 / Chapter 2.2.5.3 --- Gus protein extraction from protoplasts --- p.59 / Chapter 2.2.5.4 --- Gus assay --- p.60 / Chapter 2.2.5.5 --- MU calibration standard --- p.60 / Chapter 2.2.5.6 --- Sample assay --- p.60 / Chapter 3 --- Result --- p.61 / Chapter 3.1 --- Expression of ASN2 and ammonium assay in Arabidopsis thaliana under various stress conditions and senescence --- p.61 / Chapter 3.1.1 --- Ammonium assay of wild type seedlings under stress conditions --- p.61 / Chapter 3.1.2 --- Kinetic studies of ASN2 expression under different stresses treatments --- p.65 / Chapter 3.1.3 --- Ammonium assay of wild type seedlings under stress conditions --- p.70 / Chapter 3.2 --- NH4+ regulation on expression of ASN2 promoter --- p.73 / Chapter 3.2.1 --- The cloning ASN2 promoter --- p.73 / Chapter 3.2.1.1 --- Defining of ASN2 promoter region --- p.73 / Chapter 3.2.1.2 --- PCR amplification of ASN2 promoter from genomic sequence --- p.77 / Chapter 3.2.1.3 --- Cloning ASN2 promoter into transient gene expression vector (pBI221 vector) --- p.80 / Chapter 3.2.2 --- Transient gene expression --- p.84 / Chapter 3.2.2.1 --- Arabidopsis leave mesophyll protoplasts isolation --- p.84 / Chapter 3.2.2.2 --- Transformation and GUS expression assay --- p.87 / Chapter 3.3 --- Characterization ASN2 transgenic plants under stress conditions --- p.91 / Chapter 3.3.1 --- Construction of ASN2 transgenic plants --- p.91 / Chapter 3.3.2 --- Characterization of ASN2 transgenic plants --- p.93 / Chapter 3.3.2.1 --- Ammonium assay of ASN2 transgenic plant under different concentration of ammonium --- p.93 / Chapter 3.3.2.2 --- Ammonium assay of ASN2 transgenic plant under high light irradiance --- p.93 / Chapter 3.4 --- Characterization of mutant plants (AzaR) that showed altered ASN2 expression --- p.97 / Chapter 3.4.1 --- Phenotype of azaserine resistant mutant --- p.97 / Chapter 3.4.2 --- ASN2 expression level up-regulated in azaserine resistant mutant --- p.99 / Chapter 3.4.3 --- Checking for linkage between azaserine resistance and ASN2 overexpression --- p.101 / Chapter 3.4.4 --- Crossing the mutant with Landsberg for mapping the azaserine resistant mutant --- p.106 / Chapter 4 --- Discussion --- p.108 / Chapter 4.1 --- ASN2 may relate to ammonium metabolism --- p.108 / Chapter 4.2 --- ASN2 transgenic plants and their response under stresses conditions --- p.111 / Chapter 4.3 --- ASN2 promoter studies by transient gene expression method --- p.112 / Chapter 4.3.1 --- Identification of promoter region --- p.113 / Chapter 4.3.2 --- Isolation of protoplasts from Arabidopsis leaf --- p.114 / Chapter 4.3.3 --- Studies of ASN2 promoter transient gene expression in A thaliana protoplasts --- p.114 / Chapter 4.4 --- Azaserine Resistant Mutant --- p.115 / Chapter 4.4.1 --- Overexpression of ASN2 gene in Azaserine resistant mutant and checking for linkage --- p.115 / Chapter 4.4.2 --- Cross of Azaserine Resistant mutants with Lersberg ecotype for mapping --- p.116 / Chapter 5 --- Conclusion and prospective --- p.118 / References --- p.119 / Appendix --- p.140

by Tse Yu Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 133-138). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vi / Table of Contents --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1. --- The Plant secretory pathway --- p.2 / An overview on the secretory pathway --- p.2 / Vesicular pathways and transport vesicles --- p.4 / Chapter 2. --- Vacuolar sorting receptors --- p.6 / BP-80 and its homologues --- p.6 / RMR proteins --- p.7 / Chapter 3. --- Prevacuolar compartments --- p.8 / PVCs in mammalian and yeast cells --- p.8 / PVCs for seed protein storage vacuoles --- p.9 / PVCs for lytic vacuoles --- p.11 / Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Markers for Golgi and Prevacuolar Compartments --- p.15 / Chapter 1. --- Introduction --- p.16 / Chapter 1.1 --- Fluorescent proteins are useful tools in studying protein trafficking and subcellular localization in living cells --- p.16 / Chapter 1.2 --- Tobacco BY-2 cells --- p.18 / Chapter 1.3 --- Plant prevacuolar compartments --- p.19 / Chapter 2. --- Materials and Methods --- p.21 / Chapter 2.1 --- Construction of RFP-BP-80 and RFP-α-TIP reporters --- p.21 / Chapter 2.2 --- Construction of YFP-BP-80 and YFP-α-TIP reporters --- p.27 / Chapter 2.3 --- Construction of YFP markers for Golgi organelles --- p.32 / Chapter 2.4 --- Agrobacterium electroporation --- p.33 / Chapter 2.5 --- Transformation of tobacco BY-2 cells --- p.34 / Chapter 2.6 --- Screening of transgenic BY-2 cells expressing RFP markers --- p.35 / Chapter 2.8 --- Production of anti-BP-80 CT antibody --- p.43 / Chapter 2.9 --- Chemicals --- p.45 / Chapter 2.10 --- Primers --- p.45 / Chapter 2.11 --- Bacterial strain --- p.46 / Chapter 3. --- Results --- p.47 / Chapter 3.1 --- Generation and characterization of transgenic BY-2 cell lines expressing RFP reporters --- p.47 / Chapter 3.2 --- Generation and preliminary characterization of transgenic BY-2 cell lines expressing YFP reporters --- p.55 / Chapter 3.3 --- Confocal detection ofYFP reporters in transgenic cell lines --- p.64 / Chapter 3.4 --- Characterization of anti-BP-80 CT antibody --- p.66 / Chapter 4. --- Discussion --- p.68 / Chapter Chapter 3 --- Dynamic of Plant Prevacuolar Compartments in Transgenic Tobacco BY-2 Cells --- p.72 / Chapter 1. --- Introduction --- p.73 / Chapter 1.1 --- The plant secretory pathway --- p.73 / Chapter 1.2 --- Organelle markers in plant secretory pathway --- p.74 / Chapter 1.3 --- Markers for Lytic PVCs --- p.75 / Chapter 2. --- Materials and Methods --- p.77 / Chapter 2.1 --- Confocal immunofluorescence studies --- p.77 / Chapter 2.2 --- FM4-64 uptake study --- p.79 / Chapter 2.3 --- Brefeldin A treatment --- p.79 / Chapter 2.4 --- Wortmannin treatment --- p.80 / Chapter 2.5 --- Movement study of YFP-marked PVC --- p.82 / Chapter 3. --- Results --- p.83 / Chapter 3.1 --- Different internal organelles were labeled by two different YFP reporters --- p.83 / Chapter 3.2 --- The YFP-BP-80 reporter localized with endogenous VSR proteins --- p.86 / Chapter 3.3 --- Brefeldin A enlarged PVC organelles --- p.89 / Chapter 3.4 --- Identity of PVC-derived BFA-induced compartments --- p.99 / Chapter 3.5 --- Wortmannin induced PVCs to form small vacuoles --- p.102 / Chapter 3.6 --- PVCs are mobile organelles in living cells --- p.112 / Chapter 4. --- Discussion --- p.114 / Chapter Chapter 4 --- Summary and Future Perspectives --- p.123 / Chapter 1. --- Summary --- p.124 / The hypothesis --- p.124 / Development of three transgenic cell lines --- p.125 / Distinct organelles were marked by two different YFP reporters --- p.126 / The YFP-BP-80 reporter defined the lytic PVCs --- p.126 / Response of YFP-marked PVCs to Brefeldin A treatment --- p.127 / Response of YFP-marked PVCs to Wortmannin treatment --- p.127 / PVCs are mobile organelles in living cells --- p.129 / Chapter 2. --- Future perspectives --- p.130 / References --- p.133

by Yuan Dingyang. / "September 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 206-232). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

by Chen Li. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 136-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.