In vivo modification of the cell wall polysaccharide galactomannan of guar transformed with alpha-galactosidase gene cloned from senna
2001
Galactomannans are composed of a beta 1 to 4 mannan backbone with varying degrees of alpha 1 to 6 galactose substitutions and are found in the cell walls of legume endosperm. Like other cell wall polysaccharides, many factors controlling the metabolism of galactomannans remain to be elucidated. In the endospermous legume senna (Senna occidentalis) increased alpha-galactosidase activity has previously been observed to coincide temporarily with a decrease of the galactose content of the galactomannan. To evaluate the potential role of alpha-galactosidase for the control of the final galactose content, a alpha-galactosidase gene expressed in immature senna seeds was cloned and transformed into the related high-yielding species guar (Cyamopsis tetragonoloba). The isolated cDNA encoded a 406 amino acid protein with a calculated molecular mass of 44313 Da. The amino acid sequence was 75% identical to the galactomannan hydrolysing alpha-galactosidases from germinating guar and coffee bean. The senna alpha-galactosidase gene was inserted behind a wheat high-molecular-weight glutenin promoter in the vector employed for transformation of guar by Agrobacterium tumefaciens-mediated gene transfer. About 30% of the guar transformants produced endosperm with galactomannans where the galactose content was significantly reduced. After self-fertilization of primary transformants displaying the highest galactose reduction of the galactomannan, endosperms of R1 plants were analysed demonstrating that this property was inherited stably to the progeny and that it was 100% coupled to the presence of the senna alpha-galactosidase gene. This suggests that alpha-galactosidases can be involved in the determination of the final galactose content of endosperm galactomannans, demonstrating that cell wall polysaccharide biosynthesis can be modified in vivo.
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