Isolation and characterization of a <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> mutant that cannot utilize proline as the sole carbon and nitrogen source |
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Authors: | Huang?Sheng Bai?Xueliang Ma?Qingsheng Tang?Xianlai Email author" target="_blank">Wu?BoEmail author |
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Institution: | (1) Key Laboratory of Microbial and Plant Genetic Engineering of Ministry of Education, College of Life Science and Biotechnology, Guangxi University, 530004 Nanning, China;(2) Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization; College of Life Science and Biotechnology, Guangxi University, 530004 Nanning, China;(3) College of Life Science and Technology, Guangxi University, 530005 Nanning, China |
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Abstract: | Sinorhizobium fredii strain HN01 can use proline as the sole carbon and nitrogen source. A mutant strain GXHN100 unable to catabolize proline was screened from 6000 Tn5gusA5 random insertional mutants of S.fredii strain HN01. Sequencing analysis showed that an open read- ing frame, named pmrA (proline metabolic relative), was inserted by the Tn5gusA5. A positive clone, named pGXHN100 which containing 3.3kb foreign DNA fragment of S.fredii strain HN01, was isolated from a partial gene library of S.fredii HN01 by colony in situ hybridization. Sequence analysis showed that pGXHN100 contained the entire pmrA gene. The 3.3kb DNA fragment of pGXHN100 was cloned into a broad-host-range cosmid vector pLAFR3 to form plasmid pGXHN200 which was subsequently introduced into GXHN100 to form a complemented strain GXHN200. Plant test showed that GXHN100 was effective and no obvious changes in nitrogenase activity comparing with parental strain. But GXHN100 nodulated 2 days later on soybean and its nodulation efficiency and competitiveness were decreased. The complemented strain GXHN200 restored the nodulation efficiency and competitiveness of GXHN100 to the wild type. |
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Keywords: | Sinorhizobium fredii proline catabolism nodulation com- petitiveness |
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