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Studies of the Bifunctional Alpha-Amylase/Subtilisin Inhibitor from Barley

1RJ Henry, 2A Furtado, and 1KJ Scott

1Centre for Plant Conservation Genetics, Southern Cross University, Lismore, NSW, 2480.
Department of Biochemistry, University of Queensland, QLD.


The bifunctional alpha-amylase/subtilisin inhibitor (BASI) from barley was first characterised in the 1980s. Related proteins are found in other cereals (Fig. 1). This endosperm protein is relatively abundant suggesting a role as a storage protein. The BASI protein binds to the high pI alpha-amylases from barley and wheat (Battershell and Henry 1990) and apparently has a role in the regulation of endogenous alpha-amylase action (Henry et al.,1992,1993). The inhibition of bacterial subtilisin by the same protein may be associated with a defense role for this protein. These properties suggest that this seed storage protein has evolved multiple functions. Analysis of a wide range of cereal and grass seeds demonstrated that proteins with action against cereal alpha-amylases are restricted to wheat, barley and rye. The barley protein is more effective in alpha-amylase inhibition than the related proteins from wheat and rye (Henry et al.,1991). An ELISA test based upon a monoclonal antibody to the protein has been used to show that the level of the protein was remarkably constant in barley samples from a wide range of environments in Australia but did show genetic variation (Jarrett et al., 1997). The gene from barley has been characterised (Henry and Oono, 1991) and the protein expressed in bacteria (Jones et al.,1997). The promoter of the gene encoding the BASI protein was recently isolated and is being characterised in transgenic plants. The properties of this promoter suggest a role in the production of transgenic cereals with altered endosperm protein composition.

Biological and Industrial Applications

The following options have been proposed as roles for this protein or modified versions of the protein.

Improving the quality of flavour for wheat damaged by pre-harvest sprouting.
(protein expressed in micro-organisms)

Breeding sprouting tolerant wheat varieties
(trangenic wheat and conventional introgression)

Plant defense
(transgenic plants with pest and disease resistance)

Cereals with more consistent processing quality in variable environments
(exploits stable expression pattern)

Cereals with improved nutritional quality
(exploits high level of expression)

Promoter Analysis

The BASI promoter was isolated by inverse PCR. Functional analysis of progressive

deletions were conducted using the green fluorescent protein (gfp) reporter gene. Motifs confining seed specific expression and hormonal regulation were identified. The expression of gfp in transgenic barley and rice was compared to that with constitutive promoters.

Figure 1 . Comparison of bifunctional and alpha-amylase/subtilisin inhibitors from rice, wheat and barley (Ohtsubo and Richardson, 1992).


1. Battershell VG and Henry RJ (1990). High performance liquid chromatography of a-amylases from germinating wheat and complexes with the alpha-amylase inhibitor from barley, J. Cereal Sci. 12, 73-81.

2. Henry RJ and Oono K (1991). Amplification of a GC-rich sequence from barley by a two-step polymerase chain reaction in glycerol, Plant Molecular biology Reporter 9, 139-144.

3. Henry RJ, Battershell VG, Brennan PS and Oono K (1991). Control of wheat alpha-amylase using inhibitors from cereals J. Sci. Food Agric. 58, 281-284.

4. Henry RJ, McKinnon GE, Haak IC and Brennan PS (1992). Use of alpha-amylase inhibitors to control sprouting, Preharvest Sprouting in Cereals 1992 (Walker-Simmons MK and Ried JL Eds) American Association of Cereal Chemists, St Paul, 232-235.

5. Henry RJ, McKinnon GE, Heak IA and Brennan PS (1993). Genetic engineering of resistance to quality losses resulting from pre-harvest sprouting. Improvement of Cereal Quality by Genetic Engineering (Henry RJ and Ronalds JA Eds) Plenum, New York 129-134.

6. Jarrett SJ, Marschke RJ, Symons MH, Gibson CE, Fox GP and Henry RJ (1997) Alpha-amylase/subtilisin inhibitor levels in Australian barleys. Journal of cereal Science 25, 261-266.

7. Jones M, Vickers J, de Jersey J, Henry RJ, Symons M and Marschke R (1997). Bacterial expression of the bifunctional alpha-amylase subtilisin inhibitor from barley. J Inst

8. Brew, Jan-Feb 1997 103, 31-33.

9. Ohtsubo KI and Richardson M (1992). The amino acid sequence of a 20 k Da bifunctional subtilisin alpha-amylase inhibitor from brain of rice (Oryza sativa L) seeds. FEBS Letters 309, 68-72

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