本文選題：駱駝刺中慢生根瘤菌 + CCNWXJ12-2T��； 參考：《西北農(nóng)林科技大學(xué)》2014年博士論文

【摘要】：根瘤菌與豆科植物駱駝刺所形成的共生體系在西北干旱半干旱荒漠地區(qū)具有很好的生存能力，該共生體系能很好的在鹽堿地生存并表現(xiàn)極強(qiáng)的抗逆特性，在防風(fēng)固沙，防止沙漠化蔓延中起到及其重要的作用。駱駝刺中慢生根瘤菌Mesorhizobium alhagiXJ12-2T分離自新疆的疏葉駱駝刺，是實(shí)驗(yàn)室具有自主知識(shí)產(chǎn)權(quán)的新種。對(duì)該菌株進(jìn)行了抗逆性研究，結(jié)果發(fā)現(xiàn)該菌株具有非常強(qiáng)的抗逆特性，能夠耐受5%的NaCl，并且能在pH5~12的范圍內(nèi)良好生長(zhǎng)。為了揭示M. alhagi CCNWXJ12-2T的耐鹽分子機(jī)制，本文通過轉(zhuǎn)座子突變技術(shù)對(duì)該菌耐鹽相關(guān)功能基因進(jìn)行來了鑒定，結(jié)合全基因組測(cè)序?qū)υ摼臐B透調(diào)節(jié)網(wǎng)絡(luò)進(jìn)行了分析預(yù)測(cè)。 通過pRL27對(duì)M. alhagi CCNWXJ12-2T進(jìn)行插入誘變，建立了庫容達(dá)15000多的轉(zhuǎn)座子突變體庫，對(duì)突變體庫中的結(jié)合子進(jìn)行多次鹽敏感篩選，獲得了4株鹽敏感突變株。這4株鹽敏感突變株都對(duì)NaCl呈現(xiàn)不同程度的高度敏感性狀，并且表現(xiàn)出非常穩(wěn)定的敏感特性。生長(zhǎng)測(cè)定發(fā)現(xiàn)，在NaCl濃度為0.3mol/L的水平上，突變體Mam1、Mam2、Mam3呈現(xiàn)非常明顯的鹽敏感性狀，生長(zhǎng)量不到野生菌M. alhagi CCNWXJ12-2T生長(zhǎng)量的1/2，而突變體Mam4則尤為明顯，隨著NaCl濃度的增加呈非常明顯的生長(zhǎng)減弱趨勢(shì)；同時(shí)，這4株菌對(duì)KCl也表現(xiàn)出敏感性狀，而對(duì)LiCl、Na2SO4及蔗糖的敏感性不是很明顯，預(yù)示相關(guān)基因在耐鹽方面具有獨(dú)特功能。 運(yùn)用轉(zhuǎn)座子挽救法對(duì)轉(zhuǎn)座子側(cè)翼序列進(jìn)行了克隆，在獲得4個(gè)突變體Mam1、Mam2、Mam3和Mam4中，，Tn5分別插入到了不同的基因中，命名為xj_952、xj_3794、xj_1521和xj_274。與全基因組測(cè)序結(jié)果進(jìn)行比對(duì)，確定了突變基因在全基因組的位置并繪制了突變基因及側(cè)翼基因的物理圖譜，同時(shí)運(yùn)用多種方法對(duì)這4個(gè)突變基因的功能進(jìn)行預(yù)測(cè)及分析。xj_952基因所編碼假定蛋白MAXJ12_004699可能為一個(gè)多藥物抗性EamA家族膜轉(zhuǎn)運(yùn)蛋白；xj_3794基因編碼的蛋白為5,10-亞甲基四氫葉酸還原酶；xj_1521基因編碼的蛋白為假定外膜分泌蛋白MAXJ12_00972；xj_274基因所編碼假定蛋白MAXJ12_01324可能為�；D(zhuǎn)移酶家族的膜蛋白。 通過三親雜交，利用功能互補(bǔ)質(zhì)粒pBBR1-MCS5將全長(zhǎng)的xj_952、xj_3794、xj_1521和xj_274這4個(gè)基因?qū)氲较鄳?yīng)的鹽敏感突變株中進(jìn)行了功能互補(bǔ)驗(yàn)證。對(duì)功能互補(bǔ)菌在0.4mol/L NaCl的生長(zhǎng)情況進(jìn)行測(cè)定，結(jié)果發(fā)現(xiàn)相較于突變體，各功能互補(bǔ)菌在0.4mol/L NaCl條件下的生長(zhǎng)均得到不同程度的恢復(fù)，其中突變體Mam1、Mam3生長(zhǎng)量恢復(fù)到原菌的一半，而突變體Mam2、Mam4的生長(zhǎng)量恢復(fù)程度要小一些，恢復(fù)到原菌的1/3，表明這四個(gè)基因確實(shí)和鹽耐受性相關(guān)，在根瘤菌滲透調(diào)節(jié)及保護(hù)耐受脅迫方面發(fā)揮著作用。 采用Illumina HiSeq2000platform測(cè)序平臺(tái)對(duì)M. alhagi CCNWXJ12-2T進(jìn)行全基因組測(cè)序，測(cè)序工作由華大基因完成。對(duì)測(cè)序所得序列進(jìn)行了拼接并完成了精細(xì)圖的繪制。對(duì)獲得的基因參照蛋白庫KEGG、COG、SwissProt、TrEMBL、NR進(jìn)行功能注釋，全面分析了M. alhagi CCNWXJ12-2T全基因組序列特征，以及潛在的滲透調(diào)節(jié)相關(guān)基因。全基因組預(yù)測(cè)得到377個(gè)與滲透調(diào)節(jié)有關(guān)的基因，包括大量的參與滲透調(diào)節(jié)的轉(zhuǎn)運(yùn)系統(tǒng)及相容性溶質(zhì)合成及轉(zhuǎn)運(yùn)基因。根據(jù)已有研究結(jié)果對(duì)這些基因進(jìn)行歸類分析，繪制了該菌可能的滲透調(diào)節(jié)網(wǎng)絡(luò)。 通過我們的實(shí)驗(yàn)，在M. alhagi CCNWXJ12-2T中，首次鑒定了3個(gè)新的基因，命名為xj_952、xj_1521和xj_274，分別編碼假定蛋白MAXJ12_004699、假定外膜分泌蛋白MAXJ12_00972、假定MAXJ12_01324與該菌的耐鹽性相關(guān)，同時(shí)鑒定了一個(gè)已知編碼5,10-亞甲基四氫葉酸還原酶的基因xj_3794，也與該菌的耐鹽性有關(guān)。通過全長(zhǎng)擴(kuò)增、序列分析、功能預(yù)測(cè)、亞細(xì)胞定位及胞外多糖分析等方法從多方面系統(tǒng)的分析了突變基因的功能及與該菌耐鹽性的關(guān)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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：Q939.114

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