费氏中华根瘤菌HN01 nifR3基因的研究
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摘要
费氏中华根瘤菌(Sinorhizobium.fredii)HN01是一株快生型大豆根瘤菌,因其生长快,胞外多糖少,十分适合商业应用。固氮酶基因(nif基因)在根瘤菌固氮中起着重要的作用,本文利用自杀质粒诱变的方法对费氏中华根瘤菌HN01的固氮酶基因nifR3在结瘤、固氮中的作用进行了研究,并对其上游序列P1的启动子活性进行了测定。
我们依据根瘤菌属和中华根瘤菌属的nifR3家族保守序列设计引物,以费氏中华根瘤菌HN01的质粒为模版扩增,对其测序,获得一个完整的ORF,长1014bp,与Sinorhizobium medicae 1021的nifR3的序列在核苷酸水平上有80%的同源性,在氨基酸上有83%的同源性,命名为nifR3。同时得到其上游一段长286 bp的序列P1,在网上预测P1是启动子的最高得值为0.94。
将启动子疑似序列P1连接表达载体pLARF6-GUS,获得融合质粒pLARF6—GUSP1,以测定P1的启动子活性。利用β-Glucuronidase对P1进行了启动子活性测定。结果表明,长280 bp的P1序列具有启动子活性。
利用pK18mob构建nifR3基因的突变质粒,经三亲本接合导入HN01,获得突变菌株HNR33。通过PCR扩增,得到含有完整nifR3的片段,连接到表达载体pLAFR3上,获得互补质粒pHNBR3。以互补质粒为供体,通过三亲本接合导入突变株HNR33,获得互补株HNBR3。
通过对出发菌株HN01、突变体HNR33和互补菌株HNBR3进行生长影响的测试,发现都能正常生长在完全培养基上,达到稳定期的时间大概为30小时。当分别以硝酸盐和铵盐为唯一氮源时,突变体的生长状况与出发菌株的生长情况相似,说明nifR3基因对不同氮源利用的影响不大。通过植株试验,发现HNR33完全现瘤时间比出发菌株HN01晚1天,平均瘤重比HN01少0.082克,平均瘤数比HN01少4个,固氮酶酶活性也比野生菌株HN01的低。这表明nifR3基因与HN01的结瘤效率和固氮酶活性有关,并对结瘤有影响。
Sinorhizobium.fredii HN01 is a fast-growing Rhizobium strain that is able to form nitrogen-fixing nodules on soybean.It is suited for commercial production because its fast grower and fewer extracelluar polysaccher characteristics.The nitrogen fixation genes(nif genes)are essential for nitrogen-fixing.In this study,we detected the activity of a potential promoter region P1 upstream of the nifR3 and researched the function of nifR3 of S.fredii HN01 in symbiotic nitrogen fixation.
A 1.5 kb DNA fragment include the whole nifR3 gene and a putative promoter gerion P1 were firstly cloned by PCR method.Sequence analysis showed that nifR3 gene shares 80%and 83%identities with that of Sinorhizobiurn meliloti 1021 on nucleotide and amino level,respectively.
The putative promoter fragement P1 was fused to the gus reporter gene to determine its promoter activity.A 280-bp of the putative promoter region P1 fragment showed promoter activity by measurement ofβ-Glucuronidase activity. The suicide plasmid pK18mob was for construction of the mutant of HN01 disrupted in nifR3,and Non-polar mutant strain HNR33 was constructed.A recombinant plasmid pHNBR3 was firstly constructed by cloning the nifR3 gene into the expression vector pLAFR3.The pHNBR3 was then transformed into the mutant strain HNR33 to construct the complemental strain HNBR3.
Effects of nitrogen sources on the growth of the mutants were tested.There was little difference between the wild strain HN01 and the mutant strains HNR33 and HNBR3 when ammonium chlorite or nitrate was used as sole nitrogen source.Plant assays were carried out with HN01,HNR33 and HNBR3 to investigate their symbiotic nitrogen fixation.There were significant difference on the total number of the nodules and nitrogenase activity tested between the mutants and wild strain.On the other hand,little difference on the nodulation efficiency and the nodule weight per plant was found between them.These results suggest that the nifR3 genes are required for efficient nitrogen fixation and nodulation in HN01.
我们依据根瘤菌属和中华根瘤菌属的nifR3家族保守序列设计引物,以费氏中华根瘤菌HN01的质粒为模版扩增,对其测序,获得一个完整的ORF,长1014bp,与Sinorhizobium medicae 1021的nifR3的序列在核苷酸水平上有80%的同源性,在氨基酸上有83%的同源性,命名为nifR3。同时得到其上游一段长286 bp的序列P1,在网上预测P1是启动子的最高得值为0.94。
将启动子疑似序列P1连接表达载体pLARF6-GUS,获得融合质粒pLARF6—GUSP1,以测定P1的启动子活性。利用β-Glucuronidase对P1进行了启动子活性测定。结果表明,长280 bp的P1序列具有启动子活性。
利用pK18mob构建nifR3基因的突变质粒,经三亲本接合导入HN01,获得突变菌株HNR33。通过PCR扩增,得到含有完整nifR3的片段,连接到表达载体pLAFR3上,获得互补质粒pHNBR3。以互补质粒为供体,通过三亲本接合导入突变株HNR33,获得互补株HNBR3。
通过对出发菌株HN01、突变体HNR33和互补菌株HNBR3进行生长影响的测试,发现都能正常生长在完全培养基上,达到稳定期的时间大概为30小时。当分别以硝酸盐和铵盐为唯一氮源时,突变体的生长状况与出发菌株的生长情况相似,说明nifR3基因对不同氮源利用的影响不大。通过植株试验,发现HNR33完全现瘤时间比出发菌株HN01晚1天,平均瘤重比HN01少0.082克,平均瘤数比HN01少4个,固氮酶酶活性也比野生菌株HN01的低。这表明nifR3基因与HN01的结瘤效率和固氮酶活性有关,并对结瘤有影响。
Sinorhizobium.fredii HN01 is a fast-growing Rhizobium strain that is able to form nitrogen-fixing nodules on soybean.It is suited for commercial production because its fast grower and fewer extracelluar polysaccher characteristics.The nitrogen fixation genes(nif genes)are essential for nitrogen-fixing.In this study,we detected the activity of a potential promoter region P1 upstream of the nifR3 and researched the function of nifR3 of S.fredii HN01 in symbiotic nitrogen fixation.
A 1.5 kb DNA fragment include the whole nifR3 gene and a putative promoter gerion P1 were firstly cloned by PCR method.Sequence analysis showed that nifR3 gene shares 80%and 83%identities with that of Sinorhizobiurn meliloti 1021 on nucleotide and amino level,respectively.
The putative promoter fragement P1 was fused to the gus reporter gene to determine its promoter activity.A 280-bp of the putative promoter region P1 fragment showed promoter activity by measurement ofβ-Glucuronidase activity. The suicide plasmid pK18mob was for construction of the mutant of HN01 disrupted in nifR3,and Non-polar mutant strain HNR33 was constructed.A recombinant plasmid pHNBR3 was firstly constructed by cloning the nifR3 gene into the expression vector pLAFR3.The pHNBR3 was then transformed into the mutant strain HNR33 to construct the complemental strain HNBR3.
Effects of nitrogen sources on the growth of the mutants were tested.There was little difference between the wild strain HN01 and the mutant strains HNR33 and HNBR3 when ammonium chlorite or nitrate was used as sole nitrogen source.Plant assays were carried out with HN01,HNR33 and HNBR3 to investigate their symbiotic nitrogen fixation.There were significant difference on the total number of the nodules and nitrogenase activity tested between the mutants and wild strain.On the other hand,little difference on the nodulation efficiency and the nodule weight per plant was found between them.These results suggest that the nifR3 genes are required for efficient nitrogen fixation and nodulation in HN01.
引文
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