Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Sugarcane, a complex polyploid, may theoretically contain up to 12 alleles for a
particular gene at a single locus. The number of alleles and the extent of their
variation is of particular importance due to the potential for the exploitation of genetic
variation through breeding. Also, allelic variation has implications for the manipulation
of gene function via genetic engineering. Pyrophosphate dependent
phosphofructokinase (PFP) is considered a key regulatory enzyme involved in
sucrose biosynthesis, which may provide a target for genetic manipulation to
increase sucrose yields in sugarcane. The enzyme is composed of a regulatory
(alpha) and catalytic (beta) protein subunit encoded by the PFP-a and PFP-p genes
respectively. The PFP-p gene, which has been shown to be a single locus gene in
other plant species, was used in this study as a model for allelic variation in
sugarcane. Two main areas of investigation involved genomic and expression
analyses to further characterise the gene. Polymerase Chain Reaction (PCR) using
specific primers previously designed from conserved regions of the PFP-p gene from
different plant sources, were used to amplify across exons 10 to 12 of the sugarcane
PFP-p gene. Two PCR products, designated PFP-81/881250bp and PFP-81/881100bp
respectively, were obtained from commercial cultivars N19 and N21. Numerous
clones of the fragments were obtained and sequenced. International database
searches confirmed that both amplicons were identifiable as PFP-p. Comparative
sequence analysis indicated that the PFP-81/881250bP and PFP-81/881100bp
fragments were poorly homologous to each other, with higher regions of homology
residing in the putative exon regions (77-78%) compared to the intron regions (34-
56%). Although minor sequence variation was detected within the amplicon
populations, it was evident that two major variants of the PFP-p gene are present in
sugarcane. Southern hybridisation analysis revealed a simple banding pattern for
PFP-p. Also, there are DNA polymorph isms for the genomic regions corresponding to
the PFP-81/881250bP and PFP-81/881100bPfragments. Previous evidence indicates
that both variants are also present in the ancestral sugarcane germplasm and
maintain the same level of heterozygosity. The presence of both gene forms in the
ancestral and commercial germplasm prompts speculation that the two variants may
not segregate. This theory, together with the simple Southern hybridisation pattern obtained for PFP-p, leads to the hypothesis that the two gene forms are at separate
loci in the sugarcane genome, which may be closely linked on the same
chromosome. The expression of the variants was investigated during different stages
of sucrose accumulation in the sugarcane culm using a Reverse Transcription (RT)-
peR approach. A single, identical transcription product was isolated from these and
other selected tissues of the plant. In addition, the same transcript was obtained from
the ancestral species representatives of modern sugarcane, Saccharum officinarum
and Saccharum spontaneum. Sequence comparison of the transcribed product and
the derived exon regions of the two variants implies that the PFP-p gene represented
by the PFP-B 1/B81250bpvariant is being expressed in sugarcane while the gene form
characterised by the PFP-B1/B81100bp amplicon is silent. Northern hybridisation
analysis indicates that PFP-p is differentially expressed at different stages of sucrose
accumulation. PFP-p expression is higher in the immature culm tissue of sugarcane
and low in the mature culm, which suggests that PFP-p is highly regulated during
maturation. It is hypothesised that the PFP-p gene underwent duplication and that
one gene form was subject to accumulative mutations evolving into a pseudogene.
On the basis of present results, it can be suggested that future genetic manipulation
of PFP-p should involve the gene variant characterised by the PFP-B 1/B81250bp
fragment. / AFRIKAANSE OPSOMMING: Suikerriet, 'n komplekse poliploïede organisme, kan teoreties tot 12 allele vir 'n
spesifieke geen by 'n enkele lokus bevat. Die aantal allele en hul mate van variasie
is veral van belang, weens die potensiaal vir die benutting van hierdie genetiese
variasie deur teëling. Alleelvariasie het ook implikasies vir die manipulering van
geenfunksie via genetiese inginieurswese. Pirofosfaat-afhanklike fosfofruktokinase
(PFP) is 'n belangrike regulatoriese ensiem vir sukrose biosintese en kan geteiken
word vir genetiese manipulasie met die oog op verhoogde sukroseproduksie in
suikerriet. Die ensiem bestaan uit 'n regulatoriese (alfa) en katalitiese (beta) proteïen
subeenheid wat deur die PFP-a en PFP-~ gene respektiewelik gekodeer word. Die
PFP-~ geen, wat in ander plantspesies as 'n enkellokusgeen aangedui is, is in
hierdie studie gebruik as 'n model vir alleelvariasie in suikerriet. Die twee hoofroetes
van ondersoek wat gevolg is, behels genoom- en uitdrukkingsanalises om die geen
verder te karakteriseer. Eksons 10 tot 12 van die suikerriet PFP-~ geen is
geamplifiseer met behulp van die Polimerase Ketting Reaksie (PKR), bemiddel deur
die gebruik van spesifieke voorvoerders wat voorheen ontwerp was vanaf
gekonserveerde areas van die PFP-~ geen uit verskillende plantbronne. Twee PKR
produkte, genoem PFP-B1/B81250bPen PFP-B1/B81100bPrespektiewelik, is deur die
kommersiële kultivars N19 en N21 opgelewer. Verskeie fragmentklone is
gekonstrueer en hul DNA basisvolgorde is bepaal. Soektogte in internasionale
databasisse het bevestig dat beide amplikons PFP-~ was. Vergelykende DNA
basisvolgorde analise het aangedui dat PFP-B1/B81250bP en PFP-B1/B81100bP
fragmente swak homologie toon, terwyl 'n hoër mate van homologie in die
oënskynlike ekson areas (77-78%), vergeleke met die intronareas (34-56%) gevind
is. Alhoewel klein basisvolgordeverskille opgemerk is binne die amplikonpopulasies,
was dit duidelik dat twee hoofvariante van die PFP-~ geen in suikerriet teenwoordig
is. Southern hibridisasie analisie het 'n eenvoudige band patroon vir PFP-~ onthul.
Daar is ook DNA polimorfismes vir die genoomstreke wat met die PFP-B1/B81250bPen
PFP-B1/B81100bPfragmente ooreenstem. Vorige bewyse het aangetoon dat beide
variante ook in die voorvaderlike suikerriet kiemplasma voorkom en dat dieselfde
vlak van heterosigositeit gehandhaaf word. Die voorkoms van beide geenvorme in
die voorvaderlike asook kommersiële kiemplasmas stel voor dat hierdie twee variante miskien nie segregeer nie. Hierdie teorie, tesame met die eenvoudige
Southern hibridisasiepatroon vir PFP-p, lei tot die hipotese dat die twee geen vorme
by verskillende lokusse in die suikerrietgenoom voorkom, en dat hierdie lokusse nou
gekoppel mag wees op dieselfde chromosoom. Die uitdrukking van die variante is
ondersoek gedurende verskillende stadia van sukrose akkumulasie in die
suikerrietstingel deur van Trutranskripsie (TT)-PKR gebruik te maak. 'n Enkele,
identiese transkripsie produk is hieruit en uit ander geselekteerde plantweefsels
geïsoleer. Dieselfde transkrip is ook verkry vanaf die voorouerlike
spesieverteenwoordigers van hedendaagse suikerriet, Saccharum officinarum en
Saccharum spontaneum. 'n DNA basisvolgordevergelyking tussen die
getranskribeerde produk en die ekson-areas van die twee variante impliseer dat die
PFP-p geen verteenwoordig deur die PFP-B1/B81250bPvariant uitgedruk word in
suikerriet, terwyl die geen verteenwoordig deur die PFP-B1/B81100bpamplikon stil is.
Northern hibridisasie analise toon aan dat PFP-p verskillend uitgedruk word
gedurende verskillende stadia van sukrose akkumulasie. PFP-p uitdrukking is hoër
in die onvolwasse stamweefsel van suikerriet en laag in die volwasse stam, wat
voorstel dat PFP-p hoogs gereguleer word gedurende maturasie. Daar word
gehipotetiseer dat die PFP-p geen duplikasie ondergaan het en dat een geenvorm
onderworpe was aan akkumulerende mutasies wat deur evolusie tot 'n pseudogeen
gelei het. Dit word voorgestel, gebaseer op die huidige resultate, dat toekomstige
genetiese manipulasie van die PFP-p geen, die geenvariant gekarakteriseer deur die
PFP- B1/B81250bPfragment moet behels.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52605 |
| Date | 12 1900 |
| Creators | Reddy, Sanushka |
| Contributors | Huckett, B., Botha, F., Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 164 p. : ill. |
| Rights | Stellenbosch University |
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