JOURNAL OF MICROBIOLOGY May 2011 Vol. 31 No. 3. PCR bp BCCP. pet-28a Escherichia coli BL21 DE3 Ni-NTA

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1 JOURNAL OF MICROBIOLOGY May 2011 Vol. 31 No. 3 A ACC CT * PCR A ACC 2 42 ~ 147 bp 315 ~ 677 bp bp A 3 BC BCCP CT CT pet-28a Escherichia coli BL21 DE3 Ni-NTA CT 1. 8 mg /ml ACC CT ACC CT Q78 A Cloning of Acetyl-Coenzyme A Carboxylase Gene ACC from Rhodotorula glutinis and Prokaryotic Expression of Its CT Functional Domain Gene LI Jie-qiong WANG Li-bing LIU Yang ZHENG Shi-xue YU Zi-niu State Key Lab. of Agric. Microbiol. Natl. Engin. Res. Ctr. of Microbial Pesticides Huazhong Agric. Uni. Wuhan Abstract A full length of sequence message of acetyl-coa carboxylase ACC gene from Rhodotorula glutinis was obtained using degenerate PCR and chromosome walking methods for the first time. Sequence analysis showed that ACC gene had two introns located at 42 ~ 147 bp and 315 ~ 677 bp and the total length of coding region was bp. Secondary structure analysis of the deduced amino acid sequence revealed that it had three typical functional domains of ACC biotin carboxylase BC biotin carboxyl carrier protein BCCP and carboxyl transferase CT. cdna sequence of CT functional domain was cloned and introduced into the prokaryotic expression vector pet-28a and successfully expressed in Escherichia coli BL21 DE3 and purified using Ni-NTA resin column and obtained soluble recombinant protein at concentration of 1. 8 mg / ml. This provided a valuable material to study the function of ACC and herbicidal mechanism aiming at the function of CT. Keywords Rhodotorula glutinis ACC gene clone CT functional domain prokaryotic expression A acetyl-coa carboxylase 1 4 ACC biotin carboxylase BC A biotin carboxyl carrier protein BCCP A carboxyltransferase α -CT β -CT ACC 2009ZX B ljq @ 126. com * Tel zhengsx@ mail. hzau. edu. cn

2 3 A ACC CT 7 A BC CT Ex Taq TM Polymerase LA Taq TM Polymerase 2 BCCP RNase A TaKaRa E. Z. BC CT N. A. TM Yeast RNA Kit E. Z. N. A. TM Yeast DNA ATP Mg 2 + BC BCCP Kit Omega RevertAid TM First Strand cd- CT BCCP NA Synthesis Kit T4 DNA Fermentas A AxyPrep TM DNA Gel Extraction Kit A 1 AxyPrep TM PCR Cleanup Kit AXYGEN ACC 2 HisTrap FF1 ACC YPD LB Kan 50 μg /ml DNA RNA DNA RNA Meng Omega E. Z. N. A. TM Yeast DNA Kit 3 Acinetobacter calcoaceticus E. Z. N. A. TM Yeast RNA Kit ACC PCR ACC 6 KEGG 5. 6 Ruenwai 4 Mucor A rouxii ACC Hansenula polymorpha 40% IA QKIIEEA WGYFSV 2 IN- 3 IANNG- Rhodotorula glutinis F /FY-R QK-F /WG-R 1 PCR 70% ACC PCR 50 μl DNA 1 BC BCCP CT 3 μl 10 Ex Taq TM buffer 5 μl 2. 5 mmol /L dntps GenBank 4 μl 10 μmol /L 1 μl Ex Taq TM 0. 5 μl 5 U /μl ddh 2 O μl ACC PCR 94 5 min s 51 ACC CT 45 s 72 1 min s s 72 ACCase CT AC- 1 min min Case CT PCR PMD-18T ACC CT DH5α PCR CT GenomeWalker Universal kit ACC 7 5' Rhodotorula glutinis AP1 AP2 2 GW5-1 GW5-2 Genomewalker universal kit Escherichia coli DH5α GW5-1 /AP1 GW5-2 /AP2 PCR BL21 DE3 pet28a 1 PMD-18T TaKaRa DNA OMEGA DNA 2 4 EcoR Ⅴ Pvu Ⅱ Dra Ⅰ

3 8 31 Stu Ⅰ DNA 3 4 AP2 GW5-2 PCR 6 PCR DNA Adaptor Genome walking 4 Genome walking DNA AP1 GW5-1 3' PCR 5 1 Table 1 1 ACC Primers used in the work of ACC gene clone from Rhodotorula glutinis QK-F CAGAAGATYATYGAGGAGGC 1 WG-R ACVGAGAAGTAACCCCA 1 IN-F ATYGCBAACAACGGNATYGC 2 FY-R GAGCCTCCTCRATRATCTTCTG 2 GW3-1-1 TGATTTCGACTTCTCGGGCGAGGATGCTG 3' 1 GW3-1-2 GGAGGTACTATGCACGAGCTCAACTTC 3' 1 GW3-2-1 TCCACTCTTCAGGAGATGGTTGGTCTG 3' 2 GW3-2-2 TCTCCAGTGGGCTCATCAGGTGTCTTC 3' 2 GW3-3-1 GAAGTATGACGGCAAGGCTCCCGACGA 3' 3 GW3-3-2 CGTCGGTATGGTTGCGTGGAAGTTTGA 3' 3 GW5-1-1 AAAGGTCTCGTAGGCCCATTTTCGTAC 5' GW5-1-2 CCTTTACTGAGGCAATCCCGTTGTTAG 5' AP1 GTAATACGACTCACTATAGGGC AP2 ACTATAGGGCACGCGTGGT RT-PCR ACC CT RevertAid TM First Strand cdna Synthesis Kit PCR cdna NH-3-E-F /NH-3-H-R CT cdna NH-3-E-F CG GAATTC CCCGAGTACCCTCGAG NH-3-H-R CCC AAGCTT AGCATCCTTCCACACAAG NH-3-E-F 16 h SDS-PAGE EcoRⅠ NH-3-H-R Ni- HindⅢ NTA PMD-18T DH5α PCR 2. 1 ACC CT DNA PMD-18T-CT EcoRⅠ HindⅢ CT 2 DNA 500 bp pet-28a 1A 808 bp 1B DH5α pet-28a-ct BL21 DE3 50 μg /ml Kan LB OD 600 = mmol /L IPTG 25 2 QK-F /WG-R IN-F /FY-R PCR bp DNA NH-IW

4 3 A ACC CT 9 1 PCR ACC DNA Fig. 1 The result of cloning partial sequence of ACC gene kb from Rhodotorula glutinis by degenerate PCR A QK-F /WG-R PCR B IN-F /FY-R PCR M DL2000 Marker A The result of PCR amplification using primers QK-F / WG-R B The result of PCR amplification using primers IN- F /FY-R M DL2000 Marker A4 A 88% A 2. 2 ACC NH-IW 5' 3' ~ 147 bp 315 ~ 677 bp bp NCBI A 3 BC BCCP CT BlastX BlastX Yarrowia lipolytica GenBank YALI0C11407p A A 98% Aspergillus nidulan FGSC 2 NH-IW Fig. 2 The result of genome walking of upstream and downstream of the partial sequence NH-IW A 5' 1 EcovⅠ B 3' 1 1 StuⅠ 2 PvuⅡ C 3' 2 1 StuⅠ D 3' 3 1 DraⅠ 2 StuⅠ M DL2000 Marker A 5' region genome walking 1 using EcovⅠ library as template B The first round of 3' region genome walking 1 using StuⅠ library as template using PvuⅡ library as template C The second round of 3' region genome walking 1 using StuⅠ library as template D The third round of 3' region genome walking 1 using DraⅠ library as template 2 using StuⅠ library as template M DL2000 Marker 2. 3 ACC CT 3A 28S RNA 18S RNA 5S RNA 3 E. Z. N. A. TM Yeast RNA Kit RNA A260 /A280 RNA RNA RNA

5 10 31 cdna CT cdna bp 3B 2. 4 ACC CT pet-28a-ct 25 SDS-PAGE 1 62 ku 3 CT 4A Fig. 3 Ni SDS- PAGE 4B 1. 8 mg /ml RNA CT cdna Total RNA of Rhodotorula glutinis and amplification of CT cdna A RNA B CT cdna M DL2000 Marker A Total RNA of Rhodotorula glutinis B cdna sequence of CT domain M DL2000 Marker 4 CT SDS-PAGE Fig. 4 SDS-PAGE analysis of the expression of CT domain A SDS-PAGE 1 pet-28a /BL21 DE3 1 ~ 4 pet-28a-ct /BL21 DE3 B Ni SDS-PAGE 1 M Marker A SDS-PAGE analysis of the expression of recombinant strains 1 control strain pet-28a /BL21 DE3 1 ~ 4 recombinant strains pet- 28a-CT /BL21 DE3 Arrow represents the location of target proteins B SDS-PAGE analysis of recombinant protein purified by Ni- NTA resin 1 the purified recombinant protein M Protein marker PCR 3 A PCR ACC PCR BC BCCP CT 3 PCR Tail-PCR 6. 5 ~ 7 kb PCR 10 SON-PCR 11 SEFA-PCR 12 cdna PCR PCR cdna PCR 10 Clonetech ACC GenomeWalker Universal kit PCR

6 3 A ACC CT 11 PCR PCR PCR PCR 4 Ruenwai R Cheevadhanarak S A 13 ACC 4 BC BCCP α- CT β-ct ACC J J J. D. W. M. - acyl- ACPs ACC 14 9 J. D. W. M. ACC ACC 11 Antal Z Rascle C ACC 5 Zhang et al. Self-Formed Adaptor PCR a Simple and Efficient Method for Chromosome Walking J. Ap- 15 CT CT ACC 12 Wang SM He J Gui ZL pl Environ Microbiol ACC CT for single cell oil production J 1 Cronan JE Jr Waldrop GL. Multi-subunit acetyl-coa carboxylases J. Progress in Lipid Research Nikolau BJ Ohlrogge JB Wurtele ES. Plant biotin-containing carboxylases J. Arch Biochem Biophys Meng X Yang JM Cao YJ et al. Increasing fatty acid production in E. coli by simulating the lipid accumulation of oleaginous microorganisms J. J Ind Microbiol Biotechnol 2010 DOI / s z. Laoteng K. Overexpression of Acetyl-CoA Carboxylase Gene of Mucor rouxii Enhanced Fatty Acid Content in Hansenula polymorpha J. Mol Biotechnol Tong L. Acetyl-coenzyme A carboxylase crucial metabolic enzyme and attractive target for drug discovery J. Cell Mol Life Sci PCR PCR J Fevre M et al. Single oligonucleotide nested PCR a rapid method for the isolation of genes and their flanking regions from expressed sequence tags J. Current Genetics Ratledge C. Fatty aid biosynthesis in microorganisms being used Biochimie Davis MS Cronan JE JR. Inhibition of Escherichia coli Acetyl Coenzyme A Carboxylase by Acyl-Acyl Carrier Protein J. J Bacteriol Zhang H Tweel B Tong L. Molecular basis for the inhibition of the carboxyltransferase domain of acetyl-coenzyme A carboxylase by haloxyfop and diclofop J. Proc Natl Acad Sci USA