内生固氮菌DX120E与甘蔗互作的生理和分子生物学基础研究

发布时间：2019-04-21 21:23

【摘要】：甘蔗是重要的糖料和能源作物,生长周期长,需肥量大,尤其是氮肥。广西是甘蔗种植大省,蔗糖年产量约占全国的70%。然而广西的甘蔗种植地区大量施用氮肥,不仅成本高而且还会造成环境污染。因此,我们需要寻找一种能够减少氮肥施用的有效途径,生物固氮则是最佳的选择。目前,已有研究人员从广西主栽甘蔗品种中分离和鉴定到一批固氮菌并对部分几个固氮菌做了初步的接种试验。然而,至今尚未发现有对联合固氮菌与甘蔗互作开展比较系统研究的报道。本研究通过对从广西主栽甘蔗品种新台糖22号中分离鉴定的一株固氮菌DX120E进行了全基因组测序,并用该固氮菌接种两个不同甘蔗品种后比较该固氮菌对甘蔗生长生理特性的影响,比较了该固氮菌与固氮模式菌株PAL5的固氮能力,同时还进行了固氮菌DX120E与甘蔗组培苗互作的蛋白质组学研究,旨在探讨固氮菌DX120E促进甘蔗生长和固氮的生理和分子机制。主要研究结果如下：1.固氮菌DX120E在2个甘蔗品种(B8和GT21)的根、叶鞘和叶内均能定殖,定殖的细菌量依次为根叶鞘叶。DX120E可以从甘蔗根表面的裂隙、主根和侧根发生处及根的断裂处入侵,主要在根表面细胞间隙和细胞内大量定殖,同时也可迁移到叶片的叶肉细胞和维管束细胞中定殖；不同接种浓度下,最大定殖数量无显著差异,1×102 CFU mL-1的接种量足够侵入甘蔗并积累定殖。2.接种固氮菌DX120E可以有效促进甘蔗植株生长和对矿质营养的吸收,显著提高甘蔗植株体内的硝酸还原酶(NR)活性,同时也能在一定程度上提高植株体内谷氨酰胺合成酶(GS)活性,增加硝态氮含量,接种固氮菌DX120E还能显著提高两个甘蔗品种叶片的净光合速率(Pn)、气孔导度(Gs)和蒸腾速率(Tr)。固氮菌DX120E与固氮模式菌株PAL5比较接种试验表明两种固氮菌均具有固氮能力,并且固氮菌DX120E表现出具有比固氮菌PAL5更高的固氮效率。3.根据16SrRNA、rpoB和gyrA三个基因的克隆和测序和系统进化树的分析结果,固氮菌DX120E归属为变栖克雷伯氏菌(Klebsiella variicola)。全基因组测序的结果显示DX120E基因组包含一条环状染色体和两个质粒。其中包含了5,718,434个核苷酸,GC含量57.1%,5 172个蛋白质编码基因,25个rRNA基因,87个tRNA基因,25个假基因和2个成簇的规律间隔的短回文重复序列(CRISPR)。GOC数据库对菌株DX120E基因组进行功能注释的结果显示在已知的基因功能分类中,糖类转运与代谢、氨基酸转运与代谢和转录这三个功能所占的比例最高,分别占预测总蛋白总数的11.3%、10.4%和9.5%。4.利用双向电泳技术,共找到固氮菌DX120E与甘蔗组培苗在1/10 MS液体培养基共培养48 h后固氮菌的差异表达蛋白共22个,质谱鉴定成功18个,其中有8个上调表达,10个下调表达。鉴定到的差异蛋白质主要参与了能量、代谢(氨基酸代谢和碳水化合物代谢)、防卫反应和细胞转运这4种功能,各类功能蛋白分别占总差异蛋白质的38.9%、27.8%、22.2%和11.1%。利用ITRAQ结合质谱分析的蛋白质组学技术,共获得与固氮菌DX120E共培养48 h后甘蔗组培苗根以上部分的差异表达蛋白246个,其中上调表达240个(已知蛋白109个),下调表达6个(已知蛋白4个)。这些已知差异表达蛋白根据蛋白质的分子功能主要分成11类,其中参与防卫反应占23.9%,参与代谢反应占16.5%,参与蛋白质转化(包括蛋白质合成、折叠加工以及蛋白质水解)占12.8%,参与能量代谢以及光合作用的均占10.1%,参与细胞内转运的占6.4%,转录相关的蛋白占5.5%,信号转导相关的蛋白占4.6%,细胞结构相关的蛋白和未知分类蛋白,均占3.7%。比例最少的是细胞生长和分化,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%.
【学位授予单位】：广西大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S566.1

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