百脉根根瘤菌nod-lacZ转录融合子构建及表达分析
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摘要
根据Mesorhizobium loti基因组信息,克隆nodA、nodB、nodD等基因的启动子序列,分别与报告基因构建相应的转录融合子(nod-lacZ),并分别导入中慢生型百脉根根瘤菌MAFF303099和广谱根瘤菌NGR234(Rhizobium sp.)中,利用百脉根根的抽提物作为诱导物,检测结瘤基因的表达。结果表明:在MAFF303099菌株中,百脉根根抽提物不能诱导nodA的表达;能诱导nodB和nodD1的表达;nodD2呈组成型表达;供试的5种不同的类黄酮化合物都不能诱导nodA基因表达;在NGR234菌株中,毛地黄酮(Luteolin)能诱导nodA表达。
The promoter sequences of nodA,nodB,nodD were cloned based on the genomic information of Mesorhizobium loti MAFF303099 and fused with the reporter gene lacZ to construct the nodlacZ fusion separately.Then these fusions were separately transformed into M.loti MAFF303099 and Rhizobium sp.NGR234 to detect the expression of these nod genes after induced by Lotus japonicus root extract(RE).The results showed that there was no expression of nodA when treated with root extracts.RE induced expression of nodBand nodD1,but showed upregulation of just 2-3 times.nodD2 expressed constitutively in M.loti MAFF303099.Neither of these induced the expression of nodA in M.loti MAFF303099 induced by 5 different flavonoid compounds separately,but the expression of nodA was detected in NGR234 strain treated by Luteolin.
The promoter sequences of nodA,nodB,nodD were cloned based on the genomic information of Mesorhizobium loti MAFF303099 and fused with the reporter gene lacZ to construct the nodlacZ fusion separately.Then these fusions were separately transformed into M.loti MAFF303099 and Rhizobium sp.NGR234 to detect the expression of these nod genes after induced by Lotus japonicus root extract(RE).The results showed that there was no expression of nodA when treated with root extracts.RE induced expression of nodBand nodD1,but showed upregulation of just 2-3 times.nodD2 expressed constitutively in M.loti MAFF303099.Neither of these induced the expression of nodA in M.loti MAFF303099 induced by 5 different flavonoid compounds separately,but the expression of nodA was detected in NGR234 strain treated by Luteolin.
引文
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[2]叶竞阳,王建云,熊小波,等.紫云英类受体蛋白激酶Nip43靶蛋白的筛选与初步鉴定[J].华中农业大学学报,2016,35(3):49-53.
[3]杨倩倩,刘元,陈大松,等.蒺藜苜蓿中1个聚半乳糖醛酸酶基因突变体的筛选及共生固氮表型的初步鉴定[J].华中农业大学学报,2017,36(4):62-70.
[4]LEROUGE P,ROCHE P,FAUCHER C,et al.Symbiotic hostspecificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal[J].Nature,1990,344(6268):781-784.
[5]黄蓓,周思瑜,李荣,等.百脉根小GTPase激活蛋白的鉴定及突变体分离[J].华中农业大学学报,2017,36(2):53-58.
[6]PERRET X,STAEHELIN C,BROUGHTONW J.Molecular basis of symbiotic promiscuity[J].Mol Biol Rev,2000,64:180-201.
[7]MADDOCKS S E,OYSTON P C F.Structure and function of the LysR-type transcriptional regulator(LTTR)family proteins[J].Microbiology,2008,154:3609-3623.
[8]SUOMINEN L,LUUKKAINEN R,ROOS C,et al.Activation of the nodA promoter by the nodD genes of Rhizobium galegae induced by synthetic flavonoids or Galega orientalis root exudate[J].FEMS microbiology letters,2003,219:225-232.
[9]KANEKO T Y,NAKAMURA S,SATO E.Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti[J].DNA Res,2000,7:331-338.
[10]GIBSON D G,YOUNG L,CHUANG R Y,et al.Enzymatic assembily of DNA molecules up to several hundred kilobases[J].Nature methods,2009,6(5):343-345.
[11]王北艳,曹宁,汤晖.苜蓿中华根瘤菌电转条件的优化[J].安徽农学通报,2008(14):30-33.
[12]刘巍峰,高东,鲍晓明,等.酿酒酵母可诱导启动子功能特性的研究[J].山东大学学报,1998,33(3):345-350.
[13]KASAI-MAITA H,HIRAKAWA H,NAKAMURA Y,et al.Commonalities and differences among symbiosis islands of three Mesorhizobium loti strains[J].Microbes and environments,2013,28:275-278.
[14]KOBAYASHI H,NACIRI-GRAVEN Y,BROUGHTON W J,et al.Flavonoids induce temporal shifts in gene-expression of nod-box controlled loci in Rhizobiumsp.NGR234[J].Molecular microbiology,2004,51(2):335-347.
[15]BANFALVI Z,NIEUWKOOP A,SCHELL M,et al.Regulation of nodgene expression in Bradyrhizobium japonicum[J].Mol Gen Genet,1988,214:420-424.
[16]PECK M C,FISHER R F,LONG S R.Diverse flavonoids stimulate NodD1binding to nodgene promoters in Sinorhizobium meliloti[J].J Bacteriol,2006,188(15):5417-5427.
[17]OKKER R J,SPAINK H P,LUGTENBERG B J,et al.Mutants in the nodFEL promoter of Rhizobium leguminosarum bv.viciae reveal a role of individual nucleotides in transcriptional activation and protein binding[J].Arch Microbiol,2001,175(2):152-160.
[18]VAN RHIJN P,VANDERLEYDEN J.The Rhizobium-plant symbiosis[J].Microbiol Rev,1995,59(1):124-142.
[19]LAGUERRE G,LOUVRIER P,ALLARD M R,et al.Compatibility of rhizobial genotypes within natural populations of Rhizobium leguminosarum biovar viciae for nodulation of host legumes[J].Applied and environmental microbiology,2003,69(4):2276-2283.