Dissertation > Biological Sciences > Cell Biology > Cell morphology

Analysis of Membrane Lipids of Rhizobia under Different Phosphorus Conditions and Sequence Analysis of the SqdB Gene Required for Biosynthesis of Phosphorus-free Lipid Sulfolipid (SL)

Author ZouXiaoLin
Tutor ChenQiang; GaoJunLian
School Sichuan Agricultural University
Course Microbiology
Keywords rhizobia membrane lipids sulfolipid(SL) sqdB sequence analysis
Type Master's thesis
Year 2012
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As one of the main components of bacterial cell membrane, lipids have important physiological functions, and often are related to bacterial stress resistance in multiple conditions such as high salt, acid and drought. Under phosphorus-limiting stress environment, some rhizobia can synthesize phosphorus-free lipids to replace certain membrane phospholipids, their competitiveness to survive in the phosphorus deficient soil are enhanced. Sulfoquinovosyl diacylglycerol(SQDG,SL) is an important phosphorus-free lipids in Rhizobium which could partially substitute for Phosphatidylglycerol(PG) to constitute the cell membrane under the phosphorus-limiting conditions, it play a role to stabilize or regulate the structure and function of the membrane, and it was shown that SL may have resistance to oxidative stress.In this study,33type strains of some known rhizobial species and47rhizobia strains isolated from various legum hosts in different regions of China were used.Both membrane lipids of these rhizobia in different phosphorus conditions and the sequences of sqdB gene required for biosynthesis of SL were analysed. The major contents and results are as follows:(1) The strains were cultured under normal phosphorus conditions and phosphorus-limiting conditions. Membrane lipids were extracted and analysed by thin-layer chromatography (TLC). The membrane lipid composition characteristics of tested strains were cleared:①The main membrane lipids of the strains belonging to the Sirtorhizobium genus are Phosphatidylglycerol(PG),Phosphatidylcholin(PC),Cardiolipin(DPG), Dimethyl-Phosphatidylethanolamin(DMPE),and Phosphatidylethanolamine/Monomethyl-Phosphatidylethanolamin(PE/MMPE) and a certain amount of phosphorus-free lipids Orinthinlipid (OL) and SL under normal phosphorus conditions; and the phospholipid content including PC, PE/MMPE, PG and DPG were decreased, phosphorus-free lipids OL and SLwere increased, a large number of Diacylglyceryl trimethylhomoserine(DGTS) were syntheticed de novo under phosphorus-limiting conditions.②There is significant diversity of membrane lipids patterns due to the different types of ornithine lipid in the Rhizobium genus. The lipid composition are divided into four classes, Class Ⅰ contains ornithine lipid of S1, P1, P1, P2, class Ⅱ contains S1, P1, P2, and class Ⅲ contains S1, P1, but class Ⅳ contains S1(OL) only. The other membrane lipids composition and their changes under different phosphorus conditions were the same as that of the Sinorhizobium strains.③Strains belonging to Mesorhizobium have the ability to synthesize large quantity of OL under normal phosphorus content, and the OL was the predominant content; On the other hand, these strains seem to lack the ability to synthesize the SL.The rest of the membrane lipids and variation was the same as that of the Sinorhizobium strains.④The membrane lipids of Bradyrhizobium strains were different from the other tested rhizobial genus, and was similar under the two different phosphorus conditions, including the PE/MMPE,DPG,PC and unknown phosphorus-free lipids, while the synthesis content of PG were very little.⑤The membrane lipids of the strains nodulating with tropical woody legume of Hainan Island were very similar to the case of Rhizobium strains. Most of the strains contained multiple types of ornithine lipid. The membrane lipids of the25strains had closely relationship to the phylogenetic position, and also related to the host plant.⑥The membrane lipids of the peanut-nodulating bradyrhizobial strains isolated from Sichuan were very similar under the two different phosphorus conditions, however, the content of PG was very low.⑦Because of containing different types of ornithine lipids,the The membrane lipids of strains nodulating with pea were diverse. We divided them into3classes,2of them were same as the strains of Sinorhizobium and Rhizobium genus,and the other was a new type of membrane lipids.(2)SqdB is the most conservative gene required for biosynthesis of SL in bacteria, which encodes the enzyme catalyzed UDP-glucose to generate UDP-sulfate6-deoxyglucose. In this study, both the full-length sequences and partial sequences of the sqdB gene were analysed:①According to the sqdB sequence of Sinorhizobium meliloti1021, specific primers were designed to PCR amplification.Then the PCR product was cloned into pGEM-T Easy vector and was sequenced, four1.2kp nucleotide sequences were obtained. One complete ORF was found in each of all four sequences Their deduced amino acid sequences shows more than90%identities to the proteins encoded by published sqdB gene. The phylogenetic tree built by MEGA5.0based on the full-length sequence of sqdB was good agreement with the16S rDNA sequence analysis results. All the four strains belonged to the phylogenetic branch of Sinorhizobium genus.②According to the highly conserved regions of the sqdB, degenerate primers were designed to PCR amplify the partial sequences of sqdB of the tested strains.Then the PCR products were sequenced and nine600-700bp nucleotide sequences were obtained. Clustering results of7strains in the phylogenetic tree based on sqdB partial sequences were consistent with the16S rDNA sequence analysis. These strains were belonged to the phylogenetic branch of the Sinorhizobium or Rhizobium genus. But the other two strains identified as Mesorhizobium and Burkholderia genus were together with Rhizobium strains in the tree.The deduced amino acid sequence had similarity ranging from93%to99%with SL(UDP-sulfuric-acid-6-deoxyglucose) synthase encoded by sqdB gene from3species:Sinorhizobium meliloti, Agrobacterium radiobacter and Rhizobium leguminosarum. Clustering results of the phylogenetic tree based on amino acid sequence were in line with that of the nucleotide sequence. The9sqdB gene sequences were obtained from3known rhizobial species and some unknown rhizobial strains isolated from4kinds of legum host plants. This shows that the sqdB gene is widely distributed, and has a great diversity.

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