苜蓿根瘤菌17560细胞膜膜脂组成分析及DGTS合成基因克隆与表达

Membrane lipids analysis of a bacterial strain 17560 isolated from nodule of Alfalfa and cloning and expression of the genes for diacylglyceryl trimethylhomoserine (DGTS) biosynthesis

摘要:

【目的】分析一株分离自黑龙江省的苜蓿根瘤菌在低磷胁迫及正常磷含量条件下细胞膜脂的组成, 并从该菌中克隆和鉴定细胞膜无磷脂二酰基甘油三甲基高丝氨酸(DGTS)合成基因。【方法】分别在不同磷含量的Sherwood基本培养基中进行根瘤菌培养, 采用Bligh-Dyer方法提取细胞膜脂, 以文献报道Sinorhizobium meliloti (苜蓿中华根瘤菌)菌株1021的脂类图谱和磷脂PE、PG、PC标准品作为参照, 利用薄层层析方法分析不同磷含量条件下培养菌株的细胞膜脂组成。根据GenBank中已发表的DGTS合成基因btaA和btaB序列设计引物, 以产DGTS菌株基因组DNA为模板, 扩增btaA和btaB同源基因, 并在E. coil BL21(DE3)表达。同时检测表达菌株是否合成细胞膜无磷脂DGTS以验证基因功能。对菌株17560进行16S rRNA基因序列分析。【结果】分离自黑龙江省的苜蓿根瘤菌17560与Sinorhizobium meliloti的16S rRNA基因序列相似性高达99.8%, 但其细胞膜脂组成明显不同于参比菌株Sinorhizobium meliloti 1021的膜脂组成。在低磷胁迫条件下, 该菌株的细胞膜脂主要由OL和DGTS等无磷脂组成, 但OL的组成明显不同, 该菌株含有3种不同类型的鸟氨酸脂(OLs), 而参比菌株Sinorhizobium meliloti 1021只含有一种类型的鸟氨酸脂(OL)。在正常磷含量条件下, 该菌株的细胞膜脂主要由PE和一种未知的含氨基磷脂组成, PG与PC的含量均较少, 而参比菌株Sinorhizobium meliloti 1021的细胞膜脂主要由PE、PG与PC组成。通过PCR扩增从产DGTS 菌株17560中获得 1 913 bp DNA片段, 经序列分析发现其中有两个ORF与菌株Sinorhizobium meliloti 1021的btaA和btaB基因序列相似性均为99%。将该DNA片段克隆于pET-30a(+)得到重组质粒pLH01, 转化宿主菌获得表达菌株E. coli BL21(DE3)·pLH01, 经IPTG诱导后产生相对分子量约为45 kD和25 kD的蛋白。薄层层析验证重组菌细胞膜脂组成, 结果表明, 表达菌株E. coli BL21(DE3)·pLH01可以在IPTG诱导后合成无磷脂DGTS, 而转入空载体pET-30a(+)的阴性对照菌株E. coli BL21(DE3)·pET-30a(+)则不能合成。【结论】系统发育地位相同的苜蓿根瘤菌株的细胞膜脂组成明显不同; 苜蓿根瘤菌的细胞膜组成随培养基中的磷含量不同而变化, 低磷胁迫条件下其细胞膜脂主要由OL和DGTS等无磷脂组成; 在Sinorhizobium 膜脂中首次发现一种未知的氨基磷脂及3种不同类型的鸟氨酸脂(OLs); 从菌株17560中克隆获得２个DGTS合成基因btaA和btaB, 在大肠杆菌中成功表达, 并证实了所表达基因的功能。

Abstract:

[Objective] The aim of this study is to analyse membrane lipids composition of a rhizobial strain isolated from nodule of Alfalfa in Heilongjiang province under phosphorus-limited condition and to clone and identify the genes required for diacylglyceryl trimethylhomoserine (DGTS) biosynthesis. [Methods] The rhizobial strain was cultured in Sherwood minimal medium containing either normal or low concentrations of inorganic phosphate.The membrane lipids were extracted by Bligh-Dyer method and were analysed by thin-layer chromatography (TLC) using both the lipids of Sinorhizobium meliloti 1021 and standard samples of some phospholipid as a reference. PCR primers was designed according to the sequences of btaA and btaB in the GenBank. The PCR-amplified btaA and btaB genes were expressed in Escherichia coli BL21(DE3). The gene function was verified by testing DGTS production. 16S rRNA gene sequences of the strain 17560 was analysed. [Results] The strain 17560 isolated from Alfalfa in Heilongjiang province shares 99.8% 16S rRNA gene sequence identity with Sinorhizobium meliloti. But its lipid compositions were quite different from that of the reference strain S. meliloti 1021. Under phosphorus-limited conditions, the main membrane lipids of the strain17560 were phosphorus-free lipids, such as ornithine lipid (OL) and DGTS. Strain 17560 contain three different types of OLs meanwhile S. meliloti 1021 contain only one type of OL. Under normal phosphorus condition, the main membrane lipids of strain 17560 were phospholipid, such as phosphatidylethanolamine (PE) and one unkonw amino-containing phospholipid, while the main membrane lipids of S. meliloti 1021 are PE, phosphatidylcholine (PC) and phosphatidylglycerol (PG). BtaA and btaB genes of strain 17560 were amplified. One PCR product of 1 913 bp was obtained, which contains two ORFs sharing 99% sequence identity with btaA and btaB genes of S. meliloti 1021 respectively. This DNA fragment was cloned to pET-30a(+) vector and transferred to E. coli BL21(DE3). The proteins of 45 kD and 25 kD were detected, and TLC analysis further revealed DGTS production in the expression strain when induced with isopropyl-b-D-thiogalactoside (IPTG). [Conclusion] Membrane lipids of rhizobial strains from Alfalfa could be quite different although their phylogenetic position were the same. Membrane lipids of rhizobial strain varies with the phosphorus content in the growth medium, the main membrane lipids of the strain were phosphorus-free lipids, such as OL and DGTS under phosphorus-limiting condition. One unknown amino-containing phospholipid and three different types of OLs was found in Sinorhizobium. The btaA and btaB genes were cloned and expressed successfully in E. coli.