Genetic transformation of soybean and tobacco with β-1,3-glucanase genes

Abstract

<p>β-1,3-glucanases (also known as callases) are enzymes that hydrolyze β-1, 3-linked glucans (calloses). They are involved in various important plant physiological and developmental processes. Premature or delayed β-1,3-glucanase function in tapetal tissue is the apparent cause of male sterility in some plants. Genetic engineering has been used successfully to manipulate callase expression in tobacco and lettuce and resulted in partial to complete male sterility in the transgenic plants, while leaving female function unimpaired. The objective of this research was to alter callase expression by genetic transformation of soybean and tobacco with β-1,3-glucanase genes. Down-regulation of callase expression was carried out in soybean by introducing an anti-sense version of a soybean flower bud specific partial β-1,3-glucanase gene (SGlu7) driven by a tapetum-specific promoter TA-29. Due to insufficient funding, high cost and low efficiency of soybean transformation, this work was terminated one year later. No transgenic plants were recovered. Binary vector pLY200.1 generated in this research could be used as a generic mother vector for expressing genes in tapetal tissue. Blast search of SGlu7 in EST database of GenBank located two soybean β-1, 3-glucanase genes. Sequencing results showed that one of them, AW-Glu, contained the complete coding sequence of the callase gene. Constructs pLY500 and pLY450 were made by inserting AW-Glu after TA-29 in pLY200.1, with pLY500 having AW-Glu in sense orientation and pLY450 having AW-Glu in anti-sense orientation. pLY450 could be used to down-regulate callase expression in soybean. pLY500 was subsequently introduced into tobacco, in order to alter the timing and/or to increase the amount of callase expression, which may cause male sterility. One plant LY500-9 displayed two distinct kinds of branches, LY500-9AB and LY500-9N. Flowers from branches of LY500-9AB had stigmas higher than the anthers, while flowers from branches of LY500-9N had stigmas about the same height as the anthers. Compared with LY500-9AB or LY500-9N, non-trangenic plants had anthers significantly taller than the stigmas. The unique floral characteristics made LY500-9AB functionally male sterile. Further research is needed to investigate the cause of the functional male sterility in LY500-9AB and its relation with the transgene AW-Glu expression.</p>