兰州大学机构库 >生命科学学院
大豆慢生型根瘤菌Bradyrhizobium japonicum中一处TetR-like蛋白在共生早期阶段的功能初探
Alternative TitleStudy on the function of  TetR - like  protein of  Bradyrhizobium japonicum in the early interaction with Soybean
陈文文
Thesis Advisor魏敏
2017-04-11
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword慢生型大豆根瘤菌 重组质粒 蛋白 体外表达 调控位点
Abstract

实验室前期研究中,利用大豆慢生型根瘤菌Brdyrhizobium japonicum USDA110基因芯片技术,大豆异黄酮genistein作为刺激元,获得了全基因组表达谱,首次发现了一处染色体位点的表达独特,不但表达时期早,而且表达强度高。生物信息学分析显示,该位点中紧密排列的3个基因(bll7019-bll7021)的产物被推测为多重耐药外排泵蛋白质(Efflux pump)大家族中的成员,相邻blr7023基因的产物为TetR调控蛋白家族成员,它们可能作为操纵子单元在共生早期阶段发挥功能。基于上述研究,本实验从遗传方面和生理方面对blr7023基因编码的TetR-like蛋白在多重耐药外排泵中的调控作用、调控位点及对菌体吸附能力的影响进行了研究。根据实验目的设计了三部分实验:首先,构建重组表达质粒获得目标蛋白及电泳阻滞实验;其次,菌体抗生素(其他有害物质)抗性实验;最后,菌体吸附实验。实验结果如下:1.blr7023基因编码的调控蛋白为可溶性蛋白且可在在E. coli BL21(DE3)内稳定表达。蛋白表达量受细菌密度、诱导物(IPTG)浓度、温度以及诱导时间的影响。最佳诱导条件为:以1:100的接种量在接种瓶内前培养菌液至OD600=0.3-0.6,诱导物IPTG浓度为 0.3 mM、37℃、160 r/min诱导3 h。2.生物信息学分析以及电泳阻滞实验显示: blr7023基因编码的TetR-like蛋白的调控位点在大豆慢生型根瘤菌属的不同种内保守存在,调控结合位点(一般包含回文序列结构)位于blr7023基因翻译起始密码子上游第72位碱基起始至99位碱基结束的27个碱基的回文序列。回文序列的碱基替换影响了调控蛋白的结合。3.菌体抗生素实验显示:与野生体菌株相比,blr7023基因的缺失显著增强了大豆根瘤菌对四环素的耐受性,推测该蛋白可能负调控多重耐药外排泵结构基因bll7019-bll7021的表达。blr7023插入突变体对其他抗生素或者有毒物质的耐受性,与野生体相比,无显著变化。4、菌体吸附实验表明:blr7023基因的缺失显著降低了该根瘤菌在早期共生阶段的吸附、固着与相应宿主根部的能力,也就影响了后期结瘤、固氮等相关能力。因此,blr7023基因是在共生早期发挥重要功能的共生有利基因。综上所述,研究明确了blr7023蛋白质调控bll7019-bll7021表达的DNA结合位点,该位点包含了回文序列AAATAAACTATACCGTATATTTTCTTT。其次,blr7023基因的缺失显著增强了对四环素的耐受性, 推测该基因产物在多重耐药外排泵中为负调控。最后,blr7023的缺失降低了菌体吸附能力从而导致结瘤延迟,即该基因参与根瘤菌在共生初期阶段土壤根际的存活以及竞争性结瘤,是一个促进共生的有利基因。

Other Abstract

Bradyrhizobium japonicum, a mutualistic symbiont of soybean (Glycine max L.), widely live in the soil of soybean cultivation region over the world. The study on its role in the symbiotic nitrogen fixation is very important. Previously, a large genomic locus of B. japonicum USDA 110 has been captured in a genome-wide transcriptional screening experiment. : This genomic loci were strongly and early induced by genistein, a soybean-released isoflavonoid. Furthemore, it covers three multidrug resistance efflux pump genes (bll7019 - bll7021), Upstream of these three genes and in the opposite orientation, gene blr7023 code for a putative TetR-type regulator. Such genetic organization suggests that they likely form an operon.In the present study, the bio-function of blr7023 gene was initially addressed on the genetic and physiological leves as following: firstly, heterologous expression of blr7023 as GST fusion protein  and electrophoretic mobility shift assay (EMSA); Secondly, the susceptibility of blr7023 mutant against antibiotics (other harmful substances); finally, the soybean root  adsorption experiment . The results showed:1.blr7023 gene can be stabilized expression as soluble GST-fusion protein in E.coli BL21 (DE3), the maxmal amount of GST-fusion protein, the was obtained under the following optimized induction conditions : To 1:100 inoculation amount in the bottle  , to cultures the  bacteria density population OD600nm = 0.30-0.6, 37 ℃, the final concentration of inducer IPTG as 0.3 mM, 160 r/min and induced 3 h.2. Bioinformatics analysis showed that blr7023 gene is highly conserved among the species the  Bradyrhizobium  japonicum genus . The DNA –binding site for blr7023 protein was found to be located within the intergenic region of  bll7022 - blr7023 with a length of 27 bases.3. The intertion mutation of blr7023 specifically enhanced its susceptibility to tetracycline in comparion with wild type B.j110. However  its susceptibility to other toxic compounds was not remarkably altered.It suggested that protein of blr7023 regulated the expression of bll7019-bll7022 as a repressor.4. Bacteria adsorption experiments showed that the lack of blr7023 gene significantly reduced the symbiosis and adsorption of rhizobia in the early stage ,  the ability of fixation to corresponding hosts  roots also affected the nodules, nitrogen fixation, etc.thereforeBlr7023 gene  plays  an important function in the early symbiotic symbiotic beneficial genes.Taken together, this study identified a regulatory site of the blr7023 product is as palindrome sequence(AAATAAACTATACCGTATATTTTCTTT) that is located between bll7022and blr7023. In addition,  the mutation of blr7023 specificity enhanced its immune to tetracycline and led to the defected abosorption ability to soybean root. Finally, the deletions ofblr7023 gene alsodelay the nodulation .That the gene may participate in rhizobia in early stages of symbiosis, rhizosphere soil  survival and competitive nodulation ability. The gene blr7023 may involved in the nodulation competitiveness of B.japonicum in the early interaction with soybean.

URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221606
Collection生命科学学院
Recommended Citation
GB/T 7714
陈文文. 大豆慢生型根瘤菌Bradyrhizobium japonicum中一处TetR-like蛋白在共生早期阶段的功能初探[D]. 兰州. 兰州大学,2017.
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