核桃细菌性黑斑病菌中脂蛋白预测及其特性研究

Prediction and Characteristic Analysis of Lipoprotein in Xanthomonas arboricola pv. juglandis

【目的】脂蛋白几乎存在于所有细菌中,其通过N端脂质结构锚定在膜中,并参与细胞膜结构构建、维护细胞膜的稳定性、信号转导和转运等过程,同时在维持其生理功能、致病性和耐药性等方面发挥着重要作用。核桃细菌性黑斑病严重危害着我国核桃产业的健康发展,然而,尚未见其脂蛋白信号肽相关研究报道。【方法】本研究基于11个核桃细菌性黑斑病菌的全基因组序列,通过LipoP v1.0在线分析程序预测,从而实现对上述细菌中脂蛋白的预测。【结果】上述细菌中总共有1718个脂蛋白,所含脂蛋白范围为148~164个,所占比例为3.62%~4.02%,脂蛋白长度集中在150~450 aa,其中长度在100~200 aa的脂蛋白数量最多,氨基酸组成以丙氨酸所占比例最高,平均为14.4%~14.8%,而天冬氨酸、组氨酸、甲硫氨酸、色氨酸的含量最低。上述菌株所含脂蛋白氨基酸长度较小,推测与脂蛋白较为灵活地实现其行使功能有关;同时,对上述蛋白特性进一步开展了研究,明确脂蛋白具有1次以上跨膜结构域不多,多数脂蛋白定位在线粒体核胞外,并且在理论等电点、不稳定系数以及总平均亲水性等方面存在着一定的差异,但是总体而言上述菌株中的脂蛋白在理化性质方面具有趋同的特性,此外,少数蛋白不具有信号肽序列,不属于典型分泌蛋白,具有转运肽的脂蛋白数量较少,且预测可靠性不高。进一步对信号肽切割位点特征进行分析,发现在上述1718个脂蛋白中,A(丙氨酸)所占比例最高,在信号肽切割位点-3、-2、-1、1、2处,A(丙氨酸)、A(丙氨酸)、C(天冬氨酸)、A(丙氨酸)、P(脯氨酸)的含量最高,且氨基酸的组成在-3和-1割位点上相对比较保守,属于A-X-C类型,即丙氨酸-任意氨基酸-天冬氨酸。最后,通过遗传关系分析发现细菌性黑斑病菌中脂蛋白序列之间在具有较高同源性以及较近亲缘关系。【结论】核桃细菌性黑斑病菌中脂蛋白在理化性质、亚细胞定位以及转运肽等方面具有趋同性特征,为进一步深入开展核桃细菌性黑斑病菌中脂蛋白的研究提供了重要的理论指导,此外,通过遗传关系分析进一步明确了与细菌性黑斑病菌中脂蛋白之间的关系,为深入探索脂蛋白在细菌性黑斑病菌中的作用奠定了坚实的理论基础。同时,核桃细菌性黑斑病菌分泌系统中SPaseI和SPaseII等不同信号肽类型的外泌蛋白在同源关系上、功能类别上是否保守,是否都具有上述研究结果的(种内、种间的)独特生物学功能还有待进一步深入研究。

[Objective]Lipoprotein exists in almost all bacteria,it is anchored in the membrane through the N-terminal lipid structure,and participate in the process of cell membrane structure construction,maintenance of cell membrane stability,signal transduction and transport.At the same time,it plays an important role in maintaining its physiological functions,pathogenicity and drug resistance.As a Gram-negative bacterium,Walnut Bacterial Black Spot is a serious threat to the healthy development of walnut industry in China.However,there is no research report on its lipoprotein signal peptide.[Method]This study was based on the whole genome sequence of 11 pathogens of Xanthomonas arboricola pv. juglandis,by means of online analysis program of LipoP v1.0,so the prediction of lipoprotein in the bacteria was realized.[Result]The results showed that there were a total of 1 718 lipoproteins in these bacteria.These strains contained lipoproteins ranging from 148-164,accounting for 3.62%-4.02%.The length of lipoproteins was concentrated between 150-450 aa,and the number of lipoproteins between 100-200 aa was the largest.Alanine had the highest proportion of amino acid composition,with an average of 14.4%-14.8%,while the contents of aspartic acid,histidine,methionine,and tryptophan were the lowest. The lipoprotein amino acids contained in these strains were relatively small in length,which was inferred to be related to the more flexible realization of the function of lipoprotein.At the same time,further studies were carried out on the characteristics of these proteins,and it was clear that lipoproteins had no transmembrane domains more than once.Most lipoproteins were located outside the mitochondrial nucleus,and there were certain differences in theoretical isoelectric point,instability coefficient,and total average hydrophilicity.However,the lipoproteins in these strains had the same characteristics in physical and chemical properties.In addition,a few proteins did not have signal peptide sequences,which was not a typical secreted protein.Also,the number of lipoproteins with transit peptides was small,and the prediction reliability was not high.Further analysis of the characteristics of the signal peptide cleavage site found that among the above 1718 lipoproteins,alanine had the highest proportion.At signal peptide cleavage sites-3,-2,-1,1,2,the contents of alanine,alanine,aspartic acid,alanine,and proline were the highest,and the amino acid composition was relatively conservative at the-3 and-1 cut positions,and belonged to the A-X-C type,namely,alanineany amino acid-aspartic acid.Finally,through genetic relationship analysis,it was found that the lipoprotein sequences in the bacterial black spot disease had a higher homology and a closer relationship.According to the genetic relationship,it could be clearly divided into 6 categories.Among them,category II and IV contained the largest number of proteins,with 171 and 124 proteins,respectively,and category III contained a smaller number of proteins,only 64 proteins.[Conclusion]The above results show the convergence characteristics of X. arboricola pv. juglandis in physical and chemical properties of lipoproteins,subcellular localization,and transit a peptide had tendency fowards converge.This provides important theoretical guidance for further in-depth research on lipoproteins in X. arboricola pv. juglandis.In addition,the genetic relationship analysis has further clarified other species that have higher homology and close genetic relationship with lipoproteins in X. arboricola pv. juglandis.It has laid a solid theoretical foundation for the in-depth exploration of the role of lipoproteins in bacterial black spot disease.At the same time,further studies are needed to make clear whether the exocrine proteins of different signal peptide types such as SPaseI and SPaseII in the secretion system of X. arboricola pv.juglandis are conserved in their homology relationships and functional categories,whether they have the unique biological functions(intra-species and inter-species)with the above-mentioned research results.