【摘要】：甘蔗是重要的糖料和能源作物,生長周期長,需肥量大,尤其是氮肥。廣西是甘蔗種植大省,蔗糖年產(chǎn)量約占全國的70%。然而廣西的甘蔗種植地區(qū)大量施用氮肥,不僅成本高而且還會造成環(huán)境污染。因此,我們需要尋找一種能夠減少氮肥施用的有效途徑,生物固氮則是最佳的選擇。目前,已有研究人員從廣西主栽甘蔗品種中分離和鑒定到一批固氮菌并對部分幾個固氮菌做了初步的接種試驗。然而,至今尚未發(fā)現(xiàn)有對聯(lián)合固氮菌與甘蔗互作開展比較系統(tǒng)研究的報道。本研究通過對從廣西主栽甘蔗品種新臺糖22號中分離鑒定的一株固氮菌DX120E進行了全基因組測序,并用該固氮菌接種兩個不同甘蔗品種后比較該固氮菌對甘蔗生長生理特性的影響,比較了該固氮菌與固氮模式菌株PAL5的固氮能力,同時還進行了固氮菌DX120E與甘蔗組培苗互作的蛋白質組學研究,旨在探討固氮菌DX120E促進甘蔗生長和固氮的生理和分子機制。主要研究結果如下：1.固氮菌DX120E在2個甘蔗品種(B8和GT21)的根、葉鞘和葉內均能定殖,定殖的細菌量依次為根葉鞘葉。DX120E可以從甘蔗根表面的裂隙、主根和側根發(fā)生處及根的斷裂處入侵,主要在根表面細胞間隙和細胞內大量定殖,同時也可遷移到葉片的葉肉細胞和維管束細胞中定殖；不同接種濃度下,最大定殖數(shù)量無顯著差異,1×102 CFU mL-1的接種量足夠侵入甘蔗并積累定殖。2.接種固氮菌DX120E可以有效促進甘蔗植株生長和對礦質營養(yǎng)的吸收,顯著提高甘蔗植株體內的硝酸還原酶(NR)活性,同時也能在一定程度上提高植株體內谷氨酰胺合成酶(GS)活性,增加硝態(tài)氮含量,接種固氮菌DX120E還能顯著提高兩個甘蔗品種葉片的凈光合速率(Pn)、氣孔導度(Gs)和蒸騰速率(Tr)。固氮菌DX120E與固氮模式菌株PAL5比較接種試驗表明兩種固氮菌均具有固氮能力,并且固氮菌DX120E表現(xiàn)出具有比固氮菌PAL5更高的固氮效率。3.根據(jù)16SrRNA、rpoB和gyrA三個基因的克隆和測序和系統(tǒng)進化樹的分析結果,固氮菌DX120E歸屬為變棲克雷伯氏菌(Klebsiella variicola)。全基因組測序的結果顯示DX120E基因組包含一條環(huán)狀染色體和兩個質粒。其中包含了5,718,434個核苷酸,GC含量57.1%,5 172個蛋白質編碼基因,25個rRNA基因,87個tRNA基因,25個假基因和2個成簇的規(guī)律間隔的短回文重復序列(CRISPR)。GOC數(shù)據(jù)庫對菌株DX120E基因組進行功能注釋的結果顯示在已知的基因功能分類中,糖類轉運與代謝、氨基酸轉運與代謝和轉錄這三個功能所占的比例最高,分別占預測總蛋白總數(shù)的11.3%、10.4%和9.5%。4.利用雙向電泳技術,共找到固氮菌DX120E與甘蔗組培苗在1/10 MS液體培養(yǎng)基共培養(yǎng)48 h后固氮菌的差異表達蛋白共22個,質譜鑒定成功18個,其中有8個上調表達,10個下調表達。鑒定到的差異蛋白質主要參與了能量、代謝(氨基酸代謝和碳水化合物代謝)、防衛(wèi)反應和細胞轉運這4種功能,各類功能蛋白分別占總差異蛋白質的38.9%、27.8%、22.2%和11.1%。利用ITRAQ結合質譜分析的蛋白質組學技術,共獲得與固氮菌DX120E共培養(yǎng)48 h后甘蔗組培苗根以上部分的差異表達蛋白246個,其中上調表達240個(已知蛋白109個),下調表達6個(已知蛋白4個)。這些已知差異表達蛋白根據(jù)蛋白質的分子功能主要分成11類,其中參與防衛(wèi)反應占23.9%,參與代謝反應占16.5%,參與蛋白質轉化(包括蛋白質合成、折疊加工以及蛋白質水解)占12.8%,參與能量代謝以及光合作用的均占10.1%,參與細胞內轉運的占6.4%,轉錄相關的蛋白占5.5%,信號轉導相關的蛋白占4.6%,細胞結構相關的蛋白和未知分類蛋白,均占3.7%。比例最少的是細胞生長和分化,占2.8%。
[Abstract]:The sugar cane is an important sugar and energy crop, the growth period is long, the fertilizer needs to be large, especially the nitrogen fertilizer. Guangxi is a big province of sugarcane, and the annual output of sucrose accounts for about 70% of the whole country. However, a large amount of nitrogen fertilizer is applied in the sugarcane planting area of Guangxi, which is not only high in cost but also in environmental pollution. Therefore, we need to find an effective way to reduce the application of nitrogen fertilizer, and the biological nitrogen fixation is the best choice. At present, the researchers have isolated and identified a batch of nitrogen-fixing bacteria from the main sugarcane varieties in Guangxi, and carried out a preliminary inoculation test on some of the nitrogen-fixing bacteria. However, it has not been found that there is a systematic study on the interaction between the nitrogen-fixing bacteria and the sugarcane. An azotobacter DX120E, which was isolated and identified from the sugarcane variety of Guangxi, was sequenced and two different sugarcane varieties were inoculated with the nitrogen-fixing bacteria, and the effect of the nitrogen-fixing bacteria on the growth and physiological characteristics of the sugarcane was compared. The nitrogen fixation ability of the nitrogen-fixing bacteria and the nitrogen-fixing mode strain PAL5 is compared, and the proteomics research on the interaction between the nitrogen-fixing bacteria DX120E and the sugarcane tissue culture seedling is also carried out, and aims to explore the physiological and molecular mechanisms of the nitrogen-fixing bacteria DX120E to promote the growth and the nitrogen fixation of the sugarcane. The main results are as follows:1. The azotobacter DX120E can colonize the roots, leaves and leaves of two sugarcane varieties (B8 and GT21). The DX120E can be invaded by the fracture of the root surface of the sugarcane, the occurrence of the main root and the lateral root and the fracture of the root, mainly in the cell gap of the root surface and the large amount of colonizing in the cell, and can also be transferred to the leaf-meat cells and the vascular bundle cells of the blade to be colonized; and under different inoculation concentrations, There was no significant difference in the maximum number of colonization, and the inoculum size of 1-102 CFU-mL-1 was sufficient to invade the sugarcane and accumulate the colonization. the inoculation of the azotobacter DX120E can effectively promote the growth of the sugarcane plants and the absorption of the mineral nutrition, obviously improve the activity of the nitrate reductase (NR) in the sugarcane plant, The net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) of the leaves of two sugarcane varieties can also be significantly improved by the inoculation of the azotobacter DX120E. The comparison between the nitrogen-fixing bacteria DX120E and the nitrogen-fixing mode strain PAL5 shows that the two nitrogen-fixing bacteria have the nitrogen-fixing capacity, and the nitrogen-fixing bacteria DX120E shows the nitrogen-fixing efficiency higher than that of the azotobacter PAL5. According to the results of the cloning and sequencing of the three genes of 16SrRNA, rpoB and gyrA and the results of the phylogenetic tree analysis, the Azotobacter DX120E belongs to Klebsiella variicola. The results of full-genome sequencing showed that the DX120E genome contained an annular chromosome and two plasmids. in which 5,718,434 nucleotides were included, and the GC content was 57.1%, 5 172 protein-encoding genes,25 rRNA genes,87 tRNA genes,25 pseudogenes and two short-repeat repeat sequences (CRISPR) with regular intervals of 2 clusters. The results of functional annotation of the strain DX120E genome by the GOC database are shown in the known gene function classification, carbohydrate transport and metabolism, The proportion of the three functions of amino acid transport and metabolism and transcription was the highest, accounting for 11.3%, 10.4% and 9.5% of total total protein. A total of 22 strains of azotobacter were co-cultured with a 1/10 MS liquid culture medium by two-dimensional electrophoresis, and 18 were successfully identified by mass spectrometry, including 8 up-regulated and 10 down-regulated. The identified difference proteins were mainly involved in the four functions of energy, metabolism (amino acid metabolism and carbohydrate metabolism), defense response and cell transport. The various functional proteins accounted for 38.9%, 27.8%, 22.2% and 11.1% of the total difference protein, respectively. After 48 h co-culture with azotobacter DX120E, a total of 246 differential expression proteins were obtained by using ITRAQ combined with mass spectrometry. The expression of 240 (known protein 109) and down-regulation of 6 (4 known proteins) was reduced. These known differential expression proteins are mainly divided into 11 classes according to the molecular function of the protein, wherein the participation defense response accounts for 23.9%, the participation in the metabolic reaction is 16.5%, the protein conversion (including protein synthesis, folding processing and protein hydrolysis) accounts for 12.8%, The energy metabolism and the photosynthesis were 10.1%, 6.4% of the participating cells, 5.5% of the transcription-related proteins, 4.6% of the protein-related proteins, and 3.7% of the protein and the unknown taxin related to the cell structure. The least percentage of the cells was cell growth and differentiation, or 2.8%.
【學位授予單位】：廣西大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S566.1

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