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  • complementation  (2)
  • Granule-bound starch synthase (GBSS)  (1)
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  • 1
    ISSN: 1573-5028
    Schlagwort(e): complementation ; expression ; co-suppression ; granule-bound starch synthase ; Solanum tubersoum L. ; transgenic inheritance
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract The wild-type gene encoding granule-bound starch synthase (GBSS) is capable of both complementing the amylosefree (amf) potato mutant and inhibiting the endogenous GBSS gene expression in wild-type potato. Co-suppression of the endogenous GBSS gene, easily visualised by staining the starch with iodine, occurred when the full-size GBSS sequence (genomic), GBSS cDNA or even the mutant amf allele were introduced into the wild-type potato. Conversely, introduction of the GBSS promoter sequence alone, did not result in co-suppression in the 80 analysed transformants. Neither the orientation of the GBSS gene with respect to kanamycin resistance nor the presence of an enhancer influenced the frequency of plants showing a co-suppression phenotype. After crossing a partially complemented amf mutant with a homozygous wild-type plant, the F1 offspring segregated into plant phenotypes with normal and decreased expression of the GBSS gene. This decreased expression correlated with the presence of a linked block of five T-DNA inserts which was previously shown to be correlated with partial complementation of the amf mutant. This crossing experiment indicates that co-suppression can cause inhibition of gene expression of both inserted and endogenous wild-type GBSS genes. The frequency of partially complemented amf plants was equal to the frequency of co-suppressed wild types when a construct, with an enhancer in front of the GBSS promoter, was used (pWAM 101E). This might suggest that partial complementation of the amf genotype caused by unstable expression of the transgene can be overcome by inserting an enhancer in front of the GBSS promoter.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 2
    ISSN: 1617-4623
    Schlagwort(e): Antisense effect ; Granule-bound starch synthase (GBSS) ; Introns Promoter ; T-DNA copy number
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Inhibition of expression of specific genes by means of antisense RNA is widely used, although little information is available regarding conditions that affect the efficacy of inhibition. In this study, inhibition of granule-bound starch synthase (GBSS), a key enzyme in starch biosynthesis, is used as a model system. Eleven antisense constructs derived from the full-length GBSS cDNA, the genomic GBSS coding region (gDNA) or fragments of each of these sequences, were analysed with respect to their inhibitory effect. Introduction of full-length gDNA constructs yielded a lower percentage of transgenic clones showing complete inhibition than did introduction of the full-length cDNA constructs. This may be caused by a lower antisense binding capacity of the former due to the relatively low GC content in intron sequences present in the gDNA constructs. The presence of multiple T-DNA insertions was related to a higher degree of inhibition. Putative polyadenylation signals on the antisense strand of the GBSS gene resulted in a premature stop of transcription of some of the antisense genes, as demonstrated by the expression of smaller antisense RNA transcripts. Introduction of antisense constructs driven by the promoter of the (target) GBSS gene resulted in a higher percentage of clones with complete inhibition than introduction of antisense constructs driven by the 35S CaMV promoter. Complete antisense inhibition was achieved in 25% of the clones carrying the antisense construct pKGBA 50, which is based on the GBSS promoter and the full-length GBSS cDNA. Thus, it is concluded that the use of pKGBA50 is very suitable for the modification of the composition of potato tuber starch via antisense RNA.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 3
    ISSN: 1572-9788
    Schlagwort(e): complementation ; granule-bound starch synthase ; Solanum tuberosum L. ; expression ; transgenic inheritance ; ploidy level
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract Theamylose-free (amf) potato mutant can easily be complemented through introduction of the wild-type gene coding for granule-bound starch synthase (GBSS). After iodine staining the starch of theamf mutant is red whereas that of the wild type and the complementedamf mutant is blue. The level of complementation of selected transformants and their sexual off-spring after backcrossing withamf was investigated using sporophytic tuber cells and gametophytic microspore cells. Two diploid and two tetraploid transformants with full complementation demonstrated the expected segregation patterns of 1:1 (one active insert) or 3:1 (two independently segregating active inserts) in the microspores and in the F1 offspring based on staining of tubers. All expected genotypes in the F1 generation were found, based on microspore segregation patterns of the individual F1 plants. Two transformants with partial complementation (mixed phenotypes) were investigated. One of them, B1, was tetraploid and duplex for the GBSS insert, which had originated through mitotic doubling of the transformed diploid cells. In the F1 generation three phenotypic classes were found:amf, fully complemented and partially complemented. The latter two classes exist independently of a simplex or duplex gene status. The second transformant with partial complementation, B10, appeared to have a complex molecular composition. One cluster of five transgenes caused the partial complementation. Fully and partially complemented phenotypic classes were found after crossing B10 with theamf mutant. Indications were found that the ploidy level of the tissue in which the genes were introduced and expressed played an important role. Firstly, partial complementation was found after transformation of the diploid and not of the tetraploidamf genotypes. Secondly, the level of complementation was higher in tissue with lower ploidy levels, as illustrated by the colour of the starch inin vitro tubers (2x–4x cells) versus field-grown tubers (16x–64x).
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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