苜蓿中华根瘤菌共生固氮相关基因突变体的分离和鉴定
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摘要
为了阐明豆科植物共生反应与免疫反应的协调机制,揭示参与植物细胞侵染、定殖及固氮过程中根瘤菌基因的功能,以苜蓿中华根瘤菌Sm2011为材料,构建了约8 000株Tn5-sacB转座子插入的突变体库。利用巢式PCR检测技术,从中鉴定和筛选出nodC、nifH、smc01188和smc04024等4个特定目标基因的插入突变体及其插入位点。通过结瘤试验考察突变体共生及固氮表型,结果显示:植株接种smc01188突变体28d后,与对照植株相比,该基因突变导致根瘤固氮酶活下降,与慢生型大豆根瘤菌该基因突变体的表型类似;smc04024基因突变不影响植株生长,根瘤发育基本正常;接种nifH突变体后,植株呈缺氮表型,生长发育受阻,根瘤为白色;接种nodC突变体后植株不结瘤,说明Tn5-SacB的插入导致nifH、nodC基因功能丧失。利用PCR检测、鉴定和分离目标基因突变体是一种行之有效的方法。
Sinorhizobium meliloti strain 2011(Sm2011)was used to construct a random mutant library consisting of approximately 8 000 clones with transposon Tn5-sacBinsertion mutagenesis technique to clarify the coordination mechanism of symbiosis and immunity in the legume-rhizobium interactionand to reveal the function of rhizobium genes involved in plant cell infection,colonization and nitrogen fixation process in the nodule.We screened insertional mutants of four specific target genes including nodC,nifH,smc01188 and smc04024 from the mutant library and identified its insertion sites with nested PCR.The symbiotic and nitrogen-fixing phenotypes of mutants were examined by nodulation experiments.The result showed that the gene mutation caused a decrease in nitrogenase activity after smc01188 mutant was inoculated for 28 days compared with the control plant root nodules,which was similar to the phenotype of the slow-growing soybean rhizobium.The smc04024 gene mutation did not affect the growth of the plant,and the root nodule development was basically normal.The plant had a nitrogen-deficient phenotype with white nodule,blocked growth and development after inoculation of the nifH mutant.There was no nodulation on the plants after the inoculation of the nodC mutant,indicating that the insertion of Tn5-SacBresulted in loss of function of the nifH and nodCgenes.Detection,identification and isolation of target gene mutants with PCR is an effective method.
Sinorhizobium meliloti strain 2011(Sm2011)was used to construct a random mutant library consisting of approximately 8 000 clones with transposon Tn5-sacBinsertion mutagenesis technique to clarify the coordination mechanism of symbiosis and immunity in the legume-rhizobium interactionand to reveal the function of rhizobium genes involved in plant cell infection,colonization and nitrogen fixation process in the nodule.We screened insertional mutants of four specific target genes including nodC,nifH,smc01188 and smc04024 from the mutant library and identified its insertion sites with nested PCR.The symbiotic and nitrogen-fixing phenotypes of mutants were examined by nodulation experiments.The result showed that the gene mutation caused a decrease in nitrogenase activity after smc01188 mutant was inoculated for 28 days compared with the control plant root nodules,which was similar to the phenotype of the slow-growing soybean rhizobium.The smc04024 gene mutation did not affect the growth of the plant,and the root nodule development was basically normal.The plant had a nitrogen-deficient phenotype with white nodule,blocked growth and development after inoculation of the nifH mutant.There was no nodulation on the plants after the inoculation of the nodC mutant,indicating that the insertion of Tn5-SacBresulted in loss of function of the nifH and nodCgenes.Detection,identification and isolation of target gene mutants with PCR is an effective method.
引文
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[15]李文清,曹文刚,李豪,等.百脉根4个共生基因在水稻中的共表达及其对水稻转录组的影响[J].华中农业大学学报,2017,36(4):55-61.
[16]汪涛,冯勇,张忠明.百脉根SERKs基因家族在根瘤共生中的功能鉴定[J].华中农业大学学报,2018,37(2):9-15.
[17]ZIPFEL C.Early molecular events in PAMP-triggered immunity[J].Curr Opin Plant Biol,2009,12:414-420.
[2]赵圣博,李友国,赵斌,等.蒺藜苜蓿中CDF家族锌转运体MtMTP3的鉴定和表达调控分析[J].华中农业大学学报,2018,37(3):52-60.
[3]ARNOLD M F F,SHABAB M,PENTERMAN J,et al.Genome-wide sensitivity analysis of the microsymbiont Sinorhizobium meliloti to symbiotically important,defensin-like host peptides[J].mBio,2017,8(4):e01060.doi:https://doi.org/10.5061/dryad.32152.
[4]陈琳,余海翔,张忠明.运用Gibson克隆技术构建蒺藜苜蓿成熟根瘤的酵母AD-cDNA文库[J].华中农业大学学报,2018,37(2):23-27.
[5]GUINEL F C.Getting around the legume nodule:I.The structure of the peripheral zone in four nodule types[J].Botany,2009,87:1117-1138.
[6]MERGAERT P,NIKOVICS K,KELEMEN Z,et al.A novel family in Medicago truncatula consisting of more than 300nodule-specific genes coding for small,secreted polypeptides with conserved cysteine motifs[J].Plant Physiol,2003,132:161-173.
[7]VAN-DE-VELDE W,ZEHIROV G,SZATMARI A,et al.Plant peptides govern terminal differentiation of bacteria in symbiosis[J].Science,2010,327:1122-1126.
[8]HAAG A F,BALOBAN M,SANI M,et al.Protection of Sinorhizobiumagainst host cysteine-rich antimicrobial peptides is critical for symbiosis[J].PLoS Biol,2011,9:e1001169.
[9]年洪娟,陈丽梅,李昆志.Tn5转座突变技术在革兰氏阴性细菌分子遗传研究中的应用[J].中国生物工程杂志,2009,29(12):114-118.
[10]SHABAB M,ARNOLD M F,PENTERMAN J,et al.Disulfide cross-linking influences symbiotic activities of nodule peptide NCR247[J].PNAS,2016,113:10157-10162.
[11]GULLY D,GARGANI D,BONALDI K,et al.A peptidoglycanremodeling enzyme is critical for bacteroid differentiation in Bradyrhizobiumspp.during legume symbiosis[J].Mol Plant Microbe Interact,2016,29:447-457.
[12]BERINGER J E,BEYNON J L,BUCHANAN-WOLLASTONA V,et al.Transfer of the drug-resistance transposon Tn5to Rhizobium[J].Nature,1978,276:633-634.
[13]李碧璇,张晴,朱辉,等.百脉根小GTPase ROPs基因反转座子插入突变体的分离和鉴定[J].华中农业大学学报,2017,36(2):45-52.
[14]周向珍,朱辉.百脉根根瘤菌MAFF303099Tn5转座子插入突变体库的构建及筛选[J].华中农业大学学报,2018,37(2):16-22.
[15]李文清,曹文刚,李豪,等.百脉根4个共生基因在水稻中的共表达及其对水稻转录组的影响[J].华中农业大学学报,2017,36(4):55-61.
[16]汪涛,冯勇,张忠明.百脉根SERKs基因家族在根瘤共生中的功能鉴定[J].华中农业大学学报,2018,37(2):9-15.
[17]ZIPFEL C.Early molecular events in PAMP-triggered immunity[J].Curr Opin Plant Biol,2009,12:414-420.