Hoque, Mohammad Shamsul
Description
The availability of nitrogen in the soil is one of the main limiting factors for plant
productivity, biomass accumulation and crop yield. Well-aerated soils are relatively rich
in N03- and poor in NH4 +, whereas in anaerobic soils of irrigated or rain-fed lowland rice,
NH4 + is the most prevalent source of nitrogen. Ammonium uptake is presumed to be
controlled by at least two gene families in plants, namely AMTJ and AMT2. Each gene
family consists of one to several members, some of which...[Show more] have been cloned in
Arabidopsis, tomato and rice. As in Arabidopsis, rice is predicted to have multiple
members for the AMTJ gene family. Preliminary studies of an ammonium transporter
(OsAMTl;l) in rice (cv. Taipei 309), presented in this thesis, revealed its expression in
roots and mature leaves under different nitrogen regimes. Expression levels were
generally higher in roots than in leaves. The project was aimed at providing an insight
into rice AMT ( OsAMT) genes, their regulation and their physiological roles in rice.
A transgenic approach was undertaken to investigate the role of OsAMTl m
ammonium uptake and consequent ammonium assimilation under different nitrogen
regimes. Two expression cassettes were made using the full-length eDNA of OsAMTl;l
to have their sense or antisense expression driven by the maize ubiquitin promoter.
Transgenic lines were produced from two rice cultivars, namely Taipei 309 and Jarrah (an
Australian cultivar), by Agrobacterium-mediated transformation using these expression
cassettes mounted on the binary vector pWBVec8 that contained a hygromycin resistance
gene (hph) as the selectable marker. Only a small number of Taipei 309 transgenic lines
could be regenerated with the antisense transgene, most of which were sterile and none
showed any down-regulation of the endogenous OsAMTl;l mRNA. No antisense
transgenic lines could be regenerated from cv. Jarrah
The Ubil(l) promoter-driven OsAMTl;l sense transgene in cvs. Taipei 309 and
Jarrah increased OsAMTl;l transcript levels, which positively correlated with transgene
copy number. Under both ammonium-fed and ammonium-starved growth condtions,
ammonium-induced depolarisation of root cell plasmamembrane electrical potentials (Em)
were substantially greater in OsAMTl; 1 over-expressing transgenic lines compared to
wild type plants. These transgenic plants showed increased ammonium uptake and root
ammonium content, however, they had decreased biomass especially when grown under
high concentrations of NH4 +. A low stringency Southern blot hybridization of genomic DNA isolated from wild
type Taipei 309 with radioactively-labelled full length OsAMTl;l eDNA showed 8-10
hybridizing bands. Using a similar low stringency hybridization approach, genomic
clones of three members of the OsAMTl family, including the OsAMTl;l, were isolated.
Sequence data from these clones revealed that all three were intronless, at least in the
coding region. These represent the first genomic clones of ammonium transporters
isolated from rice. The DNA sequences of the predicted coding regions of these genes
showed 98.6%, 81% and 73% homology to that of OsAMTl; 1 from cv Nipponbare.
On the basis of the presence of several promoter signals, such as TAT A and
CAAT boxes, a 2.4 kb sequence upstream of the ATG codon from OsAMTl;l, a 2.5 kb
fragment from clone OsAMT1;2, and a 1.4 kb fragment from clone OsAMT1;3, were
selected as putative promoter regions and cloned in front of the reporter gene UidA. These
constructs were then used to transform rice cv. Taipei 309 to study the expression and
regulation of these ammonium transporter genes. Determination of the expression pattern
of GUS in resulting transgenic lines would allow the location and level of expression of
the different OsAMT genes under various developmental and environmental conditions to
be measured. Due to time constrains, results from these experiments could not be included
in this thesis.
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