construction of gene cassette harboring HMW glutenin gene of wheat driven by γ-kafirin promoter of sorghum

Modern biological tools of genetic engineering and biotechnology can allow transfer of gene(s) across crop species. The r-DNA technology has tremendous potential to transfer bread making character of bread wheat into sorghum by transferring glutenin gene(s), which can improve the visico-elastic property of the sorghum flour/dough. These genes in addition to improving quality can significantly contribute to improve the nutritional status by the addition of more protein fractions also. In the simplest approach, new HMW gluten loci may be created via transformation to bioengineer sorghum quality. For this, amplified γ-kafirin promoter (574 bp) was subcloned in pCAMBIA1304 by replacing CaMV35S promoter (ca. 800 bp) of the gus reporter gene resulting in vector pkaf-gus, where the expression of gus reporter gene is under the control of γ-kafirin promoter. In order to construct a gene cassette where HMW glu gene(s) will be under the control of γ-kafirin gene promoter, kafirin promoter was first cloned in pUC19 and then HMW gene(s) were excised from their respective vectors and cloned under the control of promoter. Finally, two gene cassettes were developed as pKaf-Dx5 and pKaf-Dy10 where expression of the HMW glu gene Dx5 (8.7 kb) and Dy10 (6.4 kb) was driven by the γ-kafirin gene promoter. Both gene cassettes are ready to clone in any vector to bioengineer sorghum by genetic transformation Acta Biol Szeged 52(2):277-282 (2008) Key WordS glutenin kafirin gene construct cloning sorghum wheat Accepted Nov 4, 2008 *Corresponding author. E-mail: [email protected] 277 Sorghum improvement can be a multidirectional program. There could be different strategies for the enhancement of economic value of the sorghum. However, lysine content enhancement and dough making quality improvement are the major areas that need urgent attention. The scope to improve the nutritional and dough quality of sorghum grain protein by employing classical plant breeding seems to be limited as only low level of variations are available in sorghum gene pool for crossing. Only two mutant high lysine genes are currently available. These are spontaneous mutant gene h1, which was initially identified in an Ethiopian line (Singh and Axtell 1973) and P 721 opaque gene which was induced with ethyl methane sulphonate (EMS) (Axtell et al. 1979). Both of these lines can be defined as “low prolamin” mutants with pleiotropic effects on other grain characteristics. Hence, it has proved difficult to incorporate the high lysine phenotype into varieties with high yield and acceptable agronomic performance and processing properties. Of all cereal grains, wheat is unique because wheat flour has the ability to form dough that exhibits the rheological properties required for the production of bread and for the wider diversity of foods. The unique properties of the wheat grain reside primarily in the gluten forming storage proteins of its endosperm. Glutenin are among the largest protein molecules in nature (Wrigley 1996) and classified as prolamins (Shewry and Halford 2002). Wheat prolamins are characterized as HMW prolamins (High molecular weight glutenin subunits, HMW-GS), S-rich prolamins (γ-gliadin, α-gliadin and B & C type of LMW-GS) and S-poor prolamins (D type of LMW glutenin). HMW-GS have been closely associated with bread making quality. After very long investigation, it was found that the glu D1 encoded HMW glutenin subunit pair 5+10 and glu 1Ax1 is associated with greater dough strength of the wheat (Shewry and Halford 2002; Altpeter et al. 2004). D’Ovidio and Anderson (1994) confirmed the role of glu D1 (Dx5+Dy10) in bread making quality of wheat. They analyzed that the y-type subunits are the main components responsible for dough making quality of the flour while x-type subunit have only minor effect. Two hypotheses have been proposed to explain the superior bread/ dough-making quality of wheat cultivars possessing subunits Dx5+Dy10 compared with those possessing subunits Dx2+Dy12. The first hypothesis considers that the additional cysteine residue present in the N-terminal domain of the Dx5 subunit plays an important role in influencing the disulphide cross-linked