Variants of the gene encoding the beta subunit of pyrophosphate dependent phosphofructokinase (PFP) and their transcriptional expression in sugarcane

[摘要] ENGLISH ABSTRACT:Sugarcane, a complex polyploid, may theoretically contain up to 12 alleles for aparticular gene at a single locus. The number of alleles and the extent of theirvariation is of particular importance due to the potential for the exploitation of geneticvariation through breeding. Also, allelic variation has implications for the manipulationof gene function via genetic engineering. Pyrophosphate dependentphosphofructokinase (PFP) is considered a key regulatory enzyme involved insucrose biosynthesis, which may provide a target for genetic manipulation toincrease 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 genesrespectively. The PFP-p gene, which has been shown to be a single locus gene inother plant species, was used in this study as a model for allelic variation insugarcane. Two main areas of investigation involved genomic and expressionanalyses to further characterise the gene. Polymerase Chain Reaction (PCR) usingspecific primers previously designed from conserved regions of the PFP-p gene fromdifferent plant sources, were used to amplify across exons 10 to 12 of the sugarcanePFP-p gene. Two PCR products, designated PFP-81/881250bp and PFP-81/881100bprespectively, were obtained from commercial cultivars N19 and N21. Numerousclones of the fragments were obtained and sequenced. International databasesearches confirmed that both amplicons were identifiable as PFP-p. Comparativesequence analysis indicated that the PFP-81/881250bP and PFP-81/881100bpfragments were poorly homologous to each other, with higher regions of homologyresiding in the putative exon regions (77-78%) compared to the intron regions (34-56%). Although minor sequence variation was detected within the ampliconpopulations, it was evident that two major variants of the PFP-p gene are present insugarcane. Southern hybridisation analysis revealed a simple banding pattern forPFP-p. Also, there are DNA polymorph isms for the genomic regions corresponding tothe PFP-81/881250bP and PFP-81/881100bPfragments. Previous evidence indicatesthat both variants are also present in the ancestral sugarcane germplasm andmaintain the same level of heterozygosity. The presence of both gene forms in theancestral and commercial germplasm prompts speculation that the two variants maynot segregate. This theory, together with the simple Southern hybridisation patternobtained for PFP-p, leads to the hypothesis that the two gene forms are at separateloci in the sugarcane genome, which may be closely linked on the samechromosome. The expression of the variants was investigated during different stagesof sucrose accumulation in the sugarcane culm using a Reverse Transcription (RT)-peR approach. A single, identical transcription product was isolated from these andother selected tissues of the plant. In addition, the same transcript was obtained fromthe ancestral species representatives of modern sugarcane, Saccharum officinarumand Saccharum spontaneum. Sequence comparison of the transcribed product andthe derived exon regions of the two variants implies that the PFP-p gene representedby the PFP-B 1/B81250bpvariant is being expressed in sugarcane while the gene formcharacterised by the PFP-B1/B81100bp amplicon is silent. Northern hybridisationanalysis indicates that PFP-p is differentially expressed at different stages of sucroseaccumulation. PFP-p expression is higher in the immature culm tissue of sugarcaneand low in the mature culm, which suggests that PFP-p is highly regulated duringmaturation. It is hypothesised that the PFP-p gene underwent duplication and thatone gene form was subject to accumulative mutations evolving into a pseudogene.On the basis of present results, it can be suggested that future genetic manipulationof PFP-p should involve the gene variant characterised by the PFP-B 1/B81250bpfragment.

[发布机构] Stellenbosch University