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【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 bpDNA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.coliBL21(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。
【Objective】 The purpose of this study was to analyze the composition of cell membrane lipids in a Rhizobium isolated from Heilongjiang Province under low-P stress and normal phosphorus conditions. The cell-free membrane lipid-free triacylglycerol trimethylhomoserine (DGTS) ) Synthetic gene. 【Method】 Rhizobium culture was carried out in Sherwood basic medium with different phosphorus contents. Bligh-Dyer method was used to extract cell membrane lipid. The lipid profile of Sinorhizobium meliloti strain 1021 and phospholipid PE, PG , PC standard as a reference, using thin-layer chromatography analysis of cell membrane lipid composition of cultured strains under different phosphorus content. Primers were designed based on the published sequences of the btA and btaB DGTS genes in GenBank. The btaA and btaB homologues were amplified using the genomic DNA of DGTS as a template and expressed in E. coli BL21 (DE3). At the same time whether the expression of bacterial cell membrane synthesis of non-phospholipid DGTS to verify gene function. Strains 17560 were subjected to 16S rRNA gene sequence analysis. 【Result】 The sequence of 16S rRNA gene from Sinorhizobium meliloti isolated from Heilongjiang Province was up to 99.8%. However, its membrane lipid composition was significantly different from that of the reference strain Sinorhizobium meliloti 1021. Under low-P stress, the cell membrane lipid of the strain mainly consisted of no phospholipids such as OL and DGTS, but the composition of OL was obviously different. The strain contained three different types of ornithine lipids (OLs), while the reference strain Sinorhizobium meliloti 1021 contains only one type of ornithine (OL). Under the condition of normal phosphorus content, the cell membrane lipid of this strain mainly consisted of PE and an unknown amino-containing phospholipid, and the contents of PG and PC were less, while the cell membrane lipid of the reference strain Sinorhizobium meliloti 1021 was mainly composed of PE, PG and PC composition. The 1 913 bp DNA fragment was obtained by PCR amplification from DGTS strain 17560. Sequence analysis showed that two of the ORFs shared 99% identity with the btaA and btaB genes of Sinorhizobium meliloti 1021. The DNA fragment was cloned into pET-30a (+) to obtain the recombinant plasmid pLH01, transformed into host strain to obtain the expression strain E. coli BL21 (DE3) .pLH01, induced by IPTG to produce proteins with relative molecular weights of about 45 kD and 25 kD. The results showed that the E. coli BL21 (DE3) .pLH01 could synthesize the phospholipid-free DGTS after induced by IPTG, and then transfected into the empty control vector pET-30a (+) negative control strain E .coli BL21 (DE3) .pET-30a (+) can not be synthesized. 【Conclusion】 The cell membrane lipid compositions of alfalfa rhizobium strains with the same phylogenetic position are obviously different. The cell membrane composition of alfalfa rhizobia varies with the phosphorus content in the medium. Under low phosphorus stress, the cell membrane lipid is mainly composed of OL and DGTS Phospholipid; An unknown aminophospholipid and three different types of ornithine lipids (OLs) were first found in Sinorhizobium membrane lipid; Two DGTS synthetic genes btaA and btaB were cloned from strain 17560, which were successful in E. coli Expression, and confirmed the function of the expressed gene.