In order to create the Male Sterile Line in tobacco, the anti-sense fragment of HSP70 gene was linked to anther specific expression promoter TA29 and the reconstructed vector was transformed into tobacco by Agrobacterium mediated transformation, and the transformants were then screened. Gus and spot blotting hybridization analysis of the transformants indicated that anti-sense fragment of HSF70 gene had been integrated into tobacco genome and expressed, thus the male sterile tobacco line was obtained. Microscope observation of anther and pollen showed that pistils of transgenic tobacco were normal, whereas anthers and pollens were fairly abortive in the same transgenic tobacco flower, comparing with pistils and stamens in control plants. The ratio of HSI：＇70 protein before and after heat shock in mitochondrial was found to be 1.39 in control tobacco plants and 1.01 in transgenic tobacco sterile lines. This is suggested that the anti-sense gene fragment of HSP70 can effectively inhibit the expression of HSP70 protein and lead to transgenic male sterility in tobacco flowers. The assay provided a new genetic engineering method for male sterility creation in plants.
It was found that the Cytoplasmic Male Sterility (CMS) line of sorghum 3197A (3A) can be induced to become fertile by heat shock treatment, whereas the maintainer line 3197B (3B) is fertile, whether it is heat shocked or not. During the pollen mother cell (PMC) stage, with heat shock treatment, the 3A line changed into fertile from sterile, and five protein bands: 70kD (HSP 70 ), 31kD, 24kD, 18kD and 16kD were found. And identical bands were also appeared in 3B line with similar treatment. By comparing the amount of protein in mitochondria during ear stage of 3A sorghum, the amount of protein increased by 2.7 times, and was almost as much as that in 3B. A comparison of the heat shock proteins (HSPs) of mitochondria in 3A and 3B lines revealed that HSPs were encoded by the nuclear DNA and transported to the mitochondria. Moreover, HSPs were more heat stable in 3B than in 3A line, and this may be related to the stability of fertility in 3B line. To demonstrate the correlation between fertility and HSPs, DNA complementary to HSP 70 was injected into ears of 3A and 3B line. It is found that under normal conditions, there was no HSP 70 mRNA in 3A line, whereas after heat shock this mRNA appeared, and the antisense cDNA could combine with the target RNA of 3B either dealt with normal temperature or heat shocked. These results are discussed within the frame of fertility versus sterility in sorghum.
[ Objective] In order to study the relation between the HSPTO gene and male sterility of plant further. [ Methods ] Anther specific expression promoter Osg6B of rice was coloned by PCR then connected with HSP70 antisense fragment to construct HSPTO antisense expression vector. The expression vector was identified by PCR experiment and enzyme digestion. [ Result] The sequence of coloned Osg6B promoter had 97% homology to the published sequence, and the cis-regulatory element in promoter area was integrated. HSP70 antisense expression vector driven by the promoter Osg6B was confired by colony PCR and enzyme digestion. [ Conclusion] The construction of expression vector would lay solid foundation for utilization of genetic engineering male sterility of plant.
