骆驼刺中慢生根瘤菌CCNWXJ12-2~T全基因组测序及耐盐相关功能基因的研究

发布时间：2018-04-13 17:28

本文选题：骆驼刺中慢生根瘤菌 + CCNWXJ12-2T　；参考：《西北农林科技大学》2014年博士论文

【摘要】：根瘤菌与豆科植物骆驼刺所形成的共生体系在西北干旱半干旱荒漠地区具有很好的生存能力，该共生体系能很好的在盐碱地生存并表现极强的抗逆特性，在防风固沙，防止沙漠化蔓延中起到及其重要的作用。骆驼刺中慢生根瘤菌Mesorhizobium alhagiXJ12-2T分离自新疆的疏叶骆驼刺，是实验室具有自主知识产权的新种。对该菌株进行了抗逆性研究，结果发现该菌株具有非常强的抗逆特性，能够耐受5%的NaCl，并且能在pH5~12的范围内良好生长。为了揭示M. alhagi CCNWXJ12-2T的耐盐分子机制，本文通过转座子突变技术对该菌耐盐相关功能基因进行来了鉴定，结合全基因组测序对该菌的渗透调节网络进行了分析预测。通过pRL27对M. alhagi CCNWXJ12-2T进行插入诱变，建立了库容达15000多的转座子突变体库，对突变体库中的结合子进行多次盐敏感筛选，获得了4株盐敏感突变株。这4株盐敏感突变株都对NaCl呈现不同程度的高度敏感性状，并且表现出非常稳定的敏感特性。生长测定发现，在NaCl浓度为0.3mol/L的水平上，突变体Mam1、Mam2、Mam3呈现非常明显的盐敏感性状，生长量不到野生菌M. alhagi CCNWXJ12-2T生长量的1/2，而突变体Mam4则尤为明显，随着NaCl浓度的增加呈非常明显的生长减弱趋势；同时，这4株菌对KCl也表现出敏感性状，而对LiCl、Na2SO4及蔗糖的敏感性不是很明显，预示相关基因在耐盐方面具有独特功能。运用转座子挽救法对转座子侧翼序列进行了克隆，在获得4个突变体Mam1、Mam2、Mam3和Mam4中，，Tn5分别插入到了不同的基因中，命名为xj_952、xj_3794、xj_1521和xj_274。与全基因组测序结果进行比对，确定了突变基因在全基因组的位置并绘制了突变基因及侧翼基因的物理图谱，同时运用多种方法对这4个突变基因的功能进行预测及分析。xj_952基因所编码假定蛋白MAXJ12_004699可能为一个多药物抗性EamA家族膜转运蛋白；xj_3794基因编码的蛋白为5,10-亚甲基四氢叶酸还原酶；xj_1521基因编码的蛋白为假定外膜分泌蛋白MAXJ12_00972；xj_274基因所编码假定蛋白MAXJ12_01324可能为酰基转移酶家族的膜蛋白。通过三亲杂交，利用功能互补质粒pBBR1-MCS5将全长的xj_952、xj_3794、xj_1521和xj_274这4个基因导入到相应的盐敏感突变株中进行了功能互补验证。对功能互补菌在0.4mol/L NaCl的生长情况进行测定，结果发现相较于突变体，各功能互补菌在0.4mol/L NaCl条件下的生长均得到不同程度的恢复，其中突变体Mam1、Mam3生长量恢复到原菌的一半，而突变体Mam2、Mam4的生长量恢复程度要小一些，恢复到原菌的1/3，表明这四个基因确实和盐耐受性相关，在根瘤菌渗透调节及保护耐受胁迫方面发挥着作用。采用Illumina HiSeq2000platform测序平台对M. alhagi CCNWXJ12-2T进行全基因组测序，测序工作由华大基因完成。对测序所得序列进行了拼接并完成了精细图的绘制。对获得的基因参照蛋白库KEGG、COG、SwissProt、TrEMBL、NR进行功能注释，全面分析了M. alhagi CCNWXJ12-2T全基因组序列特征，以及潜在的渗透调节相关基因。全基因组预测得到377个与渗透调节有关的基因，包括大量的参与渗透调节的转运系统及相容性溶质合成及转运基因。根据已有研究结果对这些基因进行归类分析，绘制了该菌可能的渗透调节网络。通过我们的实验，在M. alhagi CCNWXJ12-2T中，首次鉴定了3个新的基因，命名为xj_952、xj_1521和xj_274，分别编码假定蛋白MAXJ12_004699、假定外膜分泌蛋白MAXJ12_00972、假定MAXJ12_01324与该菌的耐盐性相关，同时鉴定了一个已知编码5,10-亚甲基四氢叶酸还原酶的基因xj_3794，也与该菌的耐盐性有关。通过全长扩增、序列分析、功能预测、亚细胞定位及胞外多糖分析等方法从多方面系统的分析了突变基因的功能及与该菌耐盐性的关系。
[Abstract]:The symbiotic system formed by thorn rhizobia and legume camel has good ability to survive in the northwest arid and semi arid desert region, the symbiotic system can be very good in saline soil and survival showed strong resistance characteristics, in the wind and sand, prevent desertification spread plays its important role in a.pseudoalhagi. Bradyrhizobium Mesorhizobium alhagiXJ12-2T isolated from Xinjiang sparsifolia is new, with independent intellectual property rights of the laboratory. The results of study on resistance, the results showed that the strains with resistance characteristics is very strong, able to withstand 5% NaCl, and can grow well in the range of pH5~12. In order to reveal M. Alhagi CCNWXJ12-2T of the molecular mechanisms of salt tolerance, this paper identified by transposon mutagenesis of genes related to salt tolerance of this bacterium, with whole genome sequencing of the bacteria in the regulation of network penetration The analysis and prediction are carried out.
Based on the M. Alhagi CCNWXJ12-2T insertion mutagenesis of pRL27, a capacity of more than 15000 transposon mutants, multiple salt sensitive screening combined with sub mutants, obtained 4 strains of salt sensitive mutant. These 4 strains of salt sensitive mutant of NaCl showed highly sensitive traits in different degree, and showed the sensitivity very stable characteristics. The growth was found in the concentration of NaCl on the level of 0.3mol/L, Mam2, mutant Mam1, Mam3 presents the salt sensitive traits is very obvious, the growth is less than M. Alhagi CCNWXJ12-2T wild mushroom growth of 1/2, while Mam4 mutant is particularly evident, with a very significant growth trend of weakening the increase of NaCl concentration at the same time;, the 4 strains of KCl also showed sensitive traits, and for LiCl, the sensitivity of Na2SO4 and sucrose is not very obvious, that gene has a unique function in salt tolerance.
Using the transposon rescue method of transposon flanking sequences were cloned in Mam2 4 mutants Mam1, Mam3, and Mam4, Tn5 were inserted into a different gene, named xj_952, xj_3794, xj_1521 and xj_274. were compared with the results of whole genome sequencing, the gene mutation in the genome position and the mapping of the physical map of the mutant gene and flanking genes, while the use of various methods for the 4 mutations were predicted and putative protein MAXJ12_004699 may be a multi drug resistance EamA family membrane transporter.Xj_952 gene encoding xj_3794 gene encoding protein analysis; 5,10- methylenetetrahydrofolate reductase; xj_1521 gene encoding protein as the putative outer membrane of secretory protein MAXJ12_00972; xj_274 gene encoding a putative protein MAXJ12_01324 membrane protein acyltransferase family.
The three pro hybridization, using functional complementation plasmid pBBR1-MCS5 full-length xj_952, xj_3794, xj_1521 and xj_274 of the 4 genes into corresponding salt sensitive mutant of functional complemention. On the function of bacteria were determined in the complementary growth of 0.4mol/L NaCl, the results show that compared with the mutants, each function complementary growth of bacteria in the 0.4mol/L NaCl conditions are different degree of recovery, the mutant Mam1, Mam3 growth back to half of the original strain, and the mutant Mam2, Mam4 growth rate of recovery to a lesser extent, restored to the original strain 1/3, suggesting that these four genes are related and salt tolerance in Rhizobium penetration play a role in regulation and protection of stress tolerant.
Using Illumina HiSeq2000platform sequencing platform for whole genome sequencing of M. Alhagi CCNWXJ12-2T, sequencing by BGI. The sequence of stitching and finished drawing fine drawing. The reference gene protein library KEGG, COG, SwissProt, TrEMBL, NR functional annotation, a comprehensive analysis of the M. Alhagi CCNWXJ12-2T the genome sequence features, as well as potential osmoregulation related genes. The genome predicted 377 and osmoregulation related genes, including a large number of transport systems involved in osmotic regulation and compatible solutes synthesis and transport genes. These genes were classified and analyzed according to the existing research results, draw the penetration of this bacterium may regulate the network.
Through our experiments, the M. Alhagi CCNWXJ12-2T, first identified 3 new genes, named xj_952, xj_1521 and xj_274 respectively, encoding a putative protein MAXJ12_004699, putative outer membrane protein secretion of MAXJ12_00972, MAXJ12_01324 and assume that the bacteria related to salt tolerance, at the same time, the identification of a known gene xj_3794 encoding 5,10- methylenetetrahydrofolate reductase, is also related to the salt tolerance of bacteria. The full-length amplification, sequence analysis, function prediction, subcellular localization and extracellular polysaccharide analysis methods from the aspects of system analysis of gene function and the relationship between salt tolerance and the bacteria.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：Q939.114

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