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《Scientia Agricultura Sinica》 1996-03
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The Cloning and Construction Study of Artificial Sterile Gene and Restore Gene

Cao Guangcheng Sun Yongru Chen Zhankuan Zhao Shimin Shu Qunfang Ma Jiangsheng Zhang Liming Li Wenbin( Institute of Genetics, Chinese Academy of Sciences, Beijing 100101; Center for Experimental Technology, Henan Agricultural Academy of Sciences)  
The cloning of pTA29,barnase and barstar as well as the construction of two plasmids: pTABNBAR55 and pTABSBAR23 were reported in this paper. pTABN-BAR55 with pTA29-barnase chimaeric gene can induce male sterility and pTABSBAR23 with pTA29-barstar chimaeric gene can restore its male fertility when transferred into the same plant. Barnase, an extracellular ribonuclease and its intracellular specific inhibitor were amplified from total DNA of Bacillus amyloliquefaciens by PCR method and cloned into pUC18 and pUC19 separately. The two genes were sequenced by Sanger's method. The cloned barstar DNA sequence precisely corresponded to that previously reported. And barnase had one-base difference at 3'-region from the two reported sequences. But peptide sequences of them were completely identical. An anther tapetum-specific expression promoter, pTA29, was cloned by PCR method and added to 5'-terminal of barnase barstar. Nos. ter was added to their 3'-terminal to enhance expression efficiency. Then we inserted a herbicide-resistant selecting marker "bar cassettee" into those two plasmids at Hind 111 site to form plasmids pTABNBAR55 and pTABSBAR23 respectively. The plant transformed with pTABNBAR55 will express barnase protein in anther tapetum cells and the plant will show male sterility while its RNase activity destroys anther tapetum. But its male fertility can be restored to this engineered male sterile plant by crossing it with the engineered fertility restore line which has been transformed by pTABSBAR23. The expression of barstar will suppress barnase's cytotoxic activity by forming a barnase/barstar inactive complex. It is greatly hopeful that the construction and transformation of pTABNBAR55 and pTABSBAR23 into various crop plants would facilitate hybrid production.
【Fund】: 国家自然科学基金
【CateGory Index】: S330
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