Browsing by Author "Reddy, Sanushka"

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    Variants of the gene encoding the beta subunit of pyrophosphate dependent phosphofructokinase (PFP) and their transcriptional expression in sugarcane
    (Stellenbosch : Stellenbosch University, 2001-12) Reddy, Sanushka; Huckett, B.; Botha, F. C.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.
    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.