共查询到10条相似文献,搜索用时 125 毫秒
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Anja G. J. Kuipers Wim J. J. Soppe Evert Jacobsen Richard G. F. Visser 《Plant molecular biology》1994,26(6):1759-1773
Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS). GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose. The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation. Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones. Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth. This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity. Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field-grown plants as compared to expression from the 35S CaMV promoter. Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content. 相似文献
3.
Formation and Deposition of Amylose in the Potato Tuber Starch Granule Are Affected by the Reduction of Granule-Bound Starch Synthase Gene Expression 总被引:15,自引:3,他引:12 下载免费PDF全文
The synthesis of amylose in amyloplasts is catalyzed by granule-bound starch synthase (GBSS). GBSS gene expression was inhibited via antisense RNA in Agrobacterium rhizogenes-transformed potato plants. Analysis of starch production and starch granule composition in transgenic tubers revealed that reduction of GBSS activity always resulted in a reduction of the production of amylose. Field experiments, performed over a 2-year period, showed that stable inhibition of GBSS gene expression can be obtained. Microscopic evaluation of iodine-stained starch granules was shown to be a sensitive system for qualitative and quantitative examination of amylose formation in starch granules of transgenic potato tubers. In plants showing inhibition of GBSS gene expression, the reduced amylose content in tuber starch was not a consequence of a lower amylose content throughout the entire starch granule. Starch granules of transgenic tubers were found to contain amylose at a percentage similar to wild-type starch in a core of varying size at the hilum of each granule. This indicated that reduced GBSS gene expression results in amylose formation in a restricted zone of the granules. The size of this zone is suggested to be dependent on the GBSS protein level. During development of the granules, the available GBSS protein is thought to become limiting, resulting in the formation of starch that lacks amylose. RNA gel blot analysis of tuber tissue showed that inhibition of GBSS gene expression resulted in a reduced GBSS mRNA level but did not affect the expression level of other starch synthesizing enzymes. Antisense RNA could only be detected in leaf tissue of the transgenic plants. 相似文献
4.
E. Flipse J. G. Huisman B. J. de Vries J. E. M. Bergervoet E. Jacobsen R. G. F. Visser 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1994,88(3-4):369-375
Granule-bound starch synthase (GBSS) catalyses the synthesis of amylose in starch granules. Transformation of a diploid amylose-free (amf) potato mutant with the gene encoding GBSS leads to the restoration of amylose synthesis. Transformants were obtained which had wild-type levels of both GBSS activity and amylose content. It proved to be difficult to increase the amylose content above that of the wild-type potato by the introduction of additional copies of the wild-type GBSS gene. Staining of starch with iodine was suitable for investigating the degree of expression of the inserted GBSS gene in transgenic amf plants. Of the 19 investigated transformants, four had only red-staining starch in tubers indicating that no complementation of the amf mutation had occured. Fifteen complemented transformants had only blue-staining starch in tubers or tubers of different staining categories (blue, mixed and red), caused either by full or partial expression of the inserted gene. Complementation was also found in the microspores. The segregation of blue- and red-staining microspores was used to analyse the inheritance of the introduced GBSS genes. A comparison of the results from microspore staining and Southern hybridisation indicated that, in three tetraploid transgenics, the gene was probably inserted before (duplex), and in all others after, chromosome doubling (simplex). The partial complementation was not due to methylation of the HPAII/MSPI site in the promoter region. Partially complemented plants had low levels of mRNA as was found when the GBSS expression levels were inhibited by anti-sense technology. 相似文献
5.
E. R. van der Leij R. G. E. Visser K. Oosterhaven D. A. M. van der Kop E. Jacobsen W. J. Feenstra 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1991,82(3):289-295
Summary
Agrobacterium rhizogenes-mediated introduction of the wild-type allele of the gene encoding granulebound starch synthase (GBSS) into the amylose-free starch mutantamf of potato leads to restoration of GBSS activity and amylose synthesis, which demonstrates thatAmf is the structural gene for GBSS. Amylose was found in columella cells of root tips, in stomatal guard cells, tubers, and pollen, while in the control experiments using only vector DNA, these tissues remained amylose free. This confirms the fact that, in potato, GBSS is the only enzyme responsible for the presence of amylose, accumulating in all starch-containing tissues. Amylose-containing transformants showed no positive correlation between GBSS activity and amylose content, which confirms that the former is not the sole regulating factor in amylose metabolism. 相似文献
6.
Shah N. I. M. Salehuzzaman Jean-Paul Vincken Marion Van De Wal Evert Jacobsen & Richard G. F. Visser 《Plant, cell & environment》1999,22(10):1311-1318
Granule-bound starch synthase I (GBSS I) is responsible for the synthesis of amylose in starch granules. A heterologous cassava GBSS I gene was tested for its ability to restore amylose synthesis in amylose-free (amf) potato mutants. For this purpose, the cassava GBSS I was equipped with different transit peptides. In addition, a hybrid containing the potato transit peptide, the N-terminal 89 amino acids of the mature potato GBSS I, and the C-terminal part of cassava GBSS I was prepared. The transgenic starches were first analysed by iodine staining. Only with the hybrid could full phenotypic complementation of the amf mutation be achieved in 13% of the plants. Most transformants showed partial complementation, but interestingly the size of the blue core was similar in all granules derived from one tuber of a given plant. The amylose content was only partially restored, up to 60% of wild-type values or potato GBSS I-complemented plants; however, the GBSS activity in these granules was similar to that found in wild-type ones. From this, and the observation that the hybrid protein (a partial potato GBSS I look-alike) performs best, it was concluded that potato and cassava GBSS I have different intrinsic properties and that the cassava enzyme is not fully adapted to the potato situation. 相似文献
7.
Role of granule-bound starch synthase in determination of amylopectin structure and starch granule morphology in potato. 总被引:5,自引:0,他引:5
Daniel C Fulton Anne Edwards Emma Pilling Helen L Robinson Brendan Fahy Robert Seale Lisa Kato Athene M Donald Peter Geigenberger Cathie Martin Alison M Smith 《The Journal of biological chemistry》2002,277(13):10834-10841
Reductions in activity of SSIII, the major isoform of starch synthase responsible for amylopectin synthesis in the potato tuber, result in fissuring of the starch granules. To discover the causes of the fissuring, and thus to shed light on factors that influence starch granule morphology in general, SSIII antisense lines were compared with lines with reductions in the major granule-bound isoform of starch synthase (GBSS) and lines with reductions in activity of both SSIII and GBSS (SSIII/GBSS antisense lines). This revealed that fissuring resulted from the activity of GBSS in the SSIII antisense background. Control (untransformed) lines and GBSS and SSIII/GBSS antisense lines had unfissured granules. Starch analyses showed that granules from SSIII antisense tubers had a greater number of long glucan chains than did granules from the other lines, in the form of larger amylose molecules and a unique fraction of very long amylopectin chains. These are likely to result from increased flux through GBSS in SSIII antisense tubers, in response to the elevated content of ADP-glucose in these tubers. It is proposed that the long glucan chains disrupt organization of the semi-crystalline parts of the matrix, setting up stresses in the matrix that lead to fissuring. 相似文献
8.
The influence of alterations in ADP-glucose pyrophosphorylase activities on starch structure and composition in potato tubers 总被引:11,自引:0,他引:11
James R. Lloyd Franziska Springer Alain Buléon Bernd Müller-Röber Lothar Willmitzer Jens Kossmann 《Planta》1999,209(2):230-238
In order to examine whether alterations in the supply of precursor molecules into the starch biosynthetic pathway affected
various characteristics of the starch, starch was isolated from potato (Solanum tuberosum L.) tubers containing reduced amounts of the enzyme ADP-glucose pyrophosphorylase (AGPase). It was found that although the
type of crystalline polymorph in the starch was not altered, the amylose content was severely reduced. In addition, amylopectin
from the transgenic plants accumulated more relatively short chains than that from control plants and the sizes of starch
granules were reduced. The starch granules from the transgenic plants contained a greater amount of granule-bound starch synthase
enzyme, which led to an increase in the maximum activity of the enzyme per unit starch tested. The K
m for ADP-glucose was, at most, only slightly altered in the transgenic lines. Potato plants containing reduced AGPase activity
were also transformed with a bacterial gene coding for AGPase to test whether this enzyme can incorporate phosphate monoesters
into amylopectin. A slight increase in phosphate contents in the starch in comparison with the untransformed control was found,
but not in comparison with starch from the line with reduced AGPase activity into which the bacterial gene was transformed.
Received: 2 February 1999 / Accepted: 25 March 1999 相似文献
9.
Transgenic potato (Solanum tuberosum L.) plants were created with sense and antisense copies of the potato D-enzyme (disproportionating enzyme; EC␣2.4.1.25) cDNA
linked to patatin and cauliflower mosaic virus 35 S promoters, and screened for D-enzyme activity in tubers. Transformants
with sense constructs mostly had wild type D-enzyme activity but two plants had only about 1% wild-type activity. Transformants
with antisense constructs had activity ranging from 90% to about 1% of wild type. Three 35 S antisense plants with very low
activity were analysed in detail. Western blot analysis showed that D-enzyme was present in greatly reduced amounts in tubers
and in leaves, whereas plastidic starch phosphorylase (EC 2.4.1.1) was unaffected. The lack of D-enzyme resulted in slow plant
growth but development was otherwise apparently normal. Furthermore, the starch content of tubers was not appreciably altered
in amount, proportion of amylose, molecular weight of debranched amylopectin, or branch chain length, despite the lack of
D-enzyme. These results do not indicate a direct requirement for D-enzyme in the synthesis and accumulation of storage starch
in tubers. The results are discussed in terms of the known reactions catalysed by D-enzyme and possible involvement of D-enzyme
in starch metabolism.
Received: 12 November 1997 / Accepted: 23 December 1997 相似文献
10.
Inhibition of the gene expression for granule-bound starch synthase I by RNA interference in sweet potato plants 总被引:3,自引:0,他引:3
Otani M Hamada T Katayama K Kitahara K Kim SH Takahata Y Suganuma T Shimada T 《Plant cell reports》2007,26(10):1801-1807
Granule-bound starch synthase I (GBSSI) is one of the key enzymes catalyzing the formation of amylose, a linear α(1,4)D-glucan
polymer, from ADP-glucose. Amylose-free transgenic sweet potato plants were produced by inhibiting sweet potato GBSSI gene expression through RNA interference. The gene construct consisting of an inverted repeat of the first exon separated
by intron 1 of GBSSI driven by the CaMV 35S promoter was integrated into the sweet potato genome by Agrobacterium tumefaciens-mediated transformation. In over 70% of the regenerated transgenic plants, the expression of GBSSI was inactivated giving rise to storage roots containing amylopectin but not amylose. Electrophoresis analysis failed to detect
the GBSSI protein, suggesting that gene silencing of the GBSSI gene had occurred. These results clearly demonstrate that amylose synthesis is completely inhibited in storage roots of sweet
potato plants by the constitutive production of the double-stranded RNA of GBSSI fragments. We conclude that RNA interference is an effective method for inhibiting gene expression in the starch metabolic
pathway. 相似文献