Induciable Male Sterility In Transgenic Plants

Finding male sterility in crops is the key point of utilization of heterosis. Since 1990, transgenic male sterility was improving. Several inducible systems for transgene expression were constructed in plant. The promoter of heat shock protein and copper-inducible system were used to control the expression of site-specific recombination enzyme gene (Cre) in our research. By excision of blocking fragment between anther-specific promoter (Osg6B) and RNase gene (barnase), effects of induciable male sterility system was investigated in these transgenic plants.1. First, the promoter (Genbank Number: AF544399) of heat shock protein Gmhspl7.5C was cloned from soybean genome. The Gus reporter gene driven by Gmhspl7.5C promoter was transferred into tobacco. The result of histochemical stain and quantitative fluorometric Gus assays showed that after twice heat shock treatment (42'C 2h), leaves and stems of transgenic tobacco displayed markedly Gus activity, and Gus activity was 5-6 times of that before heat shock treatment in leaves. Second, a DNA fragment of CaMV35S-Gus franked by two same orientation loxp sites could be excised from the transgenic tobacco genome by Cre expression under the control of heat shock promoter. The result showed that 41% of DNA fragment of CaMV35S-Gus could be excised after heat shock treatment.2. The heat-inducible, site-specific recombination system was used to control the expression of barnase gene in the anther of transgenic tobacco. The binary vector containing Cre gene under control of Gmhspl7.5C promoter and a blocking fragment franked by two loxp sites between promoter Osg6B and barnase gene was constructed. The result of investigation for shape of flower and vigor of pollen after heat shock treatment showed filament markedly shortened, anther withered, fertile pollen were few, the most of pollen couldn't germinate and yield of mature fruit decreased significantly. Transgenic lines were semi or the most male sterility.3. The heat-inducible, site-specific recombination system was used to control the expression of barnase gene in the anther of transgenic wheat. The vector with barnase gene driven by Osg6B promoter, the vector with Cre gene driven by heat shock promoter, the vector with blocking fragment between Osg6B promoter and barnase gene, the vector with barstar gene (a inhibitor for barnase gene) driven by Osg6B promoter were transferred into wheat by biolistic method. The result of staining of pollen showed different levels of male sterility in TO generation transgenic lines transferred with barnase gene driven by Osg6B promoter. Most of pollen in T1 generation transgenic lines with barnase and Cre gene were sterile after heat shock treatment. The results of investigation for T1 transgenic lines with barnase and Cre gene showed yield of mature fruit of transgenic lines induced in seeds and seedling was less slightly than that of transgenic lines no induced or wild wheat.4. The copper-inducible, site-specific recombination system was used to control the expression of barnase gene in the anther of transgenic wheat. The Cre gene driven by copper-inducible system and vector with blocking fragment between Osg6B promoter and barnase gene were transferred into wheat by biolistic method. The result of staining of pollen showed some pollen in T1 generation transgenic lines with barnase and Cre gene were sterile after induction with copper (50 M CuSO4) solution. The results of investigation for T1 transgenic lines with barnase and Cre gene showed yield of mature fruit of transgenic lines induced by copper solution decreased slightly.5. A promoter of transcription factor (AtMYB 103) to control product of plant pollen was cloned from Arabidopsis. The Gus reporter gene driven by AtMYB 103 promoter was transferred into tobacco. The result of histochemical staining showed that AtMYB 103 promoter showed activity in anther and pollen but not in other tissue of flower.