Nucleotide sequence of a wheat (Triticum aestivum L.) cDNA clone encoding the waxy protein

Abstract

The waxy mutation in maize is characterised by the complete loss of amylase in the starch fraction of the kernel [1,2,3]. In addition, there is a significant reduction in the amount of granule-bound starch synthase activity in the mutant [2] which correlates with the absence of a protein of 60 kDa, which in wild-type kernels is the most abundant protein associated with starch granules [4,5]. For this reason, it has been assumed that the 60 kDa waxy protein is the granule-bound starch synthase. Unequivocal evidence for this is lacking and in pea, evidence suggests that another protein, only distantly related to the waxy protein, is the granule-bound starch synthase [6]. It remains to be seen whether this is the case in cereals. The waxy locus has been cloned from a number of plants including maize [ 3 ], rice [ 7 ], barley [ 8 ] and potato [9]. We report here the isolation of a fulllength cDNA clone for the waxy protein of hexaploid wheat and present the complete nucleotide sequence.A cDNA library of was constructed in 2gtl0 from poly(A) + RNA isolated from wheat grains harvested at 20 days post anthesis from the hexaploid variety Chinese Spring. Isolation of RNA, selection of poly(A) + RNA and cDNA synthesis were carried out according to manufacturer's protocols (Amersham). After addition of linkers the cDNAs were ligated into the Eco RI site of 2gtl0 and packaged to produce 1.5 x 10 5 recombinants per/~g poly(A) + RNA. The library was screened for waxy protein clones by hybridising plaque lifts with a barley waxy protein cDNA (pcwx27 [4]) and 9 clones with inserts that ranged in size from 0.5 to 4.4 kb were isolated and plaque-purified. The insert from a single cDNA clone of 2.2 kb was subcloned into pUC18 for further analysis since northern blots of wheat RNA hybridised with pcwx27 allowed the waxy protein mRNA size to be estimated at 2.2-2.3 kb (data not shown). The plasmid clone containing the 2.2 kb insert was designated pcSS22.The complete nucleotide sequence of the cDNA insert of pcS $22 was determined by dideoxy chain termination sequencing [ 10] of Bal 31 deletions and restriction fragments cloned into pUC18. Synthetic oligonucleotide primers to the waxy cDNA were also used. Staden programs were used for sequence assembly [11] and the U W G C G programs for sequence analysis. TheThe nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X57233.

Identification of the major starch synthase in the soluble fraction of potato tubers.

“…The dendrogram was generated using the PILEUP program (Devereux et al, 1984). The identities of the proteins are as follows: Agro GS, Agrobacterium glycogen synthase (Uttaro et al, 1990); Ecoli GS, €scherichia coli glycogen synthase (Kumar et al, 1986); Bsub GS, Bacillus subtilis glycogen synthase (Kiel et al, 1994); maize GBSSl (Klosgen et al, 1986); rice GBSSI (Okagaki, 1992); wheat GBSSI (Clark et al, 1991); barley GBSSI (Rohde et al, 1988); cassava GBSSI (Salehuzzaman et al, 1993); potato GBSSI (Visser et al, 1989); pea GBSSI (Dry et al, 1992); potato SSll (Edwards et al, 1995); pea SSll (Dry et al, 1992); rice SSS, rice soluble starch synthase (Baba et al, 1993); and potato SSlll (this study).…”

Discrete Forms of Amylose Are Synthesized by Isoforms of GBSSI in Pea[W]

“…The accession numbers for the plants described in Figure 2B are as follows: barley (Hordeum vulgare), X07932 (Rohde et al, 1988); wheatI (Triticum aestivum), X57233 (Clark et al, 1991); maize (Zea mays), X03935 (Klosgen et al, 1986); sorghum (Sorghum bicolor), Q43134 (Y.C. Hsing, unpublished data); rice (Oryza sativa), X62134 (R.J. Okayaki, unpublished data); bean (Phaseolus vulgaris), AB029546 (N. Isono, K. Nozaki, H. Ito, H. Matsui, and M. Honma, un-…”

Photoperiodic Control of Carbon Distribution during the Floral Transition inArabidopsis

“…It was originally identified in maize (Zea mays) kernels as the product of the waxy gene, and biochemical and genetic studies have shown that GBSS is responsible for the synthesis of the linear glycan (amylose) in starch (Nelson and Pan, 1995;Ball et al, 1998;Denyer et al 2001). Genes encoding the orthologous protein have been isolated from many different plant species, such as potato (Solanum tuberosum) (Dry et al, 1992;van der Steege et al, 1992), pea (Pisum sativum) (Dry et al, 1992), barley (Hordeum vulgare) (Rohde et al, 1988), wheat (Triticum aestivum) (Clark et al, 1991), snapdragon (Antirrhinum majus; Mérida et al, 1999), and Arabidopsis (Tenorio et al, 2003). Although the analysis of mutants has demonstrated that GBSS is responsible for the synthesis of amylose in storage organs of diverse plants (Smith et al, 1997;Zeeman et al, 2010), no GBSS mutant involved in the accumulation of transitory starch has been thoroughly characterized to date.…”