http / /xuebao. jxau. edu. cn. orfh79 orfh79 pet - 28a - orfh79 pgex5x orfh79

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1 http / /xuebao. jxau. edu. cn Acta Agriculturae Universitatis Jiangxiensis E - mail ndxb7775@ sina. com orfh / / orfh79 orfh79 pet - 28a - orfh79 pgex5x orfh79 BL21 DE3 IPTG PET - 28a - orfh79 ORFH79 pgex5x orfh79 ORFH79 N GST orfh79 Q943 S511 A Expression of a Mitochondrial Gene orfh79 from the CMS-Honglian Rice Inhibiting the Growth of Escherichia coli PENG Xiao-jue 1 2 ZHANG Jian 1 ZHU You-lin 1 WANG Kun 2 LI Shao-bo 1 LI Shao-qing 2 ZHU Ying-guo 2 1. Key Laboratory of Molecular Biology and Gene Engineering College of Life Science Nanchang University Nanchang China 2. Key MOE for Plant Developmental Biology College of Life Science Wuhan University Wuhan China Abstract orfh79 is a mitochondrial gene responsible for the cotyplasm male sterility of Honglian rice. To study the molecular mechanism of this gene the sequence of orfh79 gene was cloned into pet - 28a and pgex5x - 2 vectors respectively and then expressed in BL21 DE3 strain. The results showed that weakly expressed native ORFH79 protein that induced from the recombinant PET - 28a - orfh79 strongly inhibits the growth of E. coli. However the expression recombinant ORFH79 protein which fuses a GST protein in N - terminal in Escherichia coli doesn t impair the growth of E. coli. These results may provide a clue to get some information about the mechanism on cytoplasmic male sterility of Honglian rice. Key words rice cytoplasim male sterile orfh79 prokaryotic expression growth of E. coli cytoplasmic male sterility CMS CB CJJ E - mail 163. com

2 3 orfh CMS CMS CMS 14 CMS 2 T Urf13 Urf13 13 ku BmT - methomyl T BmT - methomyl 3 orf138 4 orf79 5 orf522 6 CMS 7 atp6 orfh79 coxi orfh79 orfh79 8 orfh79 orfh pet - 28a pgex5x - 2 DH5α BL21 DE T4 - DNA Takara X - gene 1. 3 PCR orfh79 Nco1 BamH1 BamH1 Xho1 PCR Nco1 5' GCC CCA TGG CAA ATC TGC TCC GAT GGC TC 3' BamH1 5' GCC GGATCCATGACA AATCTGCTCCGATGGCTC3' BamH1 5'GCCGGATCCATGACA AATCTGCTCCGATGGCTC3' Xhol1 5'GC- CCTCGAGTTACTTAGGAAAGACACG3' 25 μl dntp 0. 5 μl 10 mmol /L 200 μl /L 10 Buffer 2. 5 μl Taq 0. 2 μl 5 μ /L 1 μl 1 μl 10 μmol /L 1 μl ddh 2 O μl 94 5 min s s s min10 g /L PCR PCR 5 h 1. 4 Nco1 BamH1 pet - 28a T4 orfh79 pet - 28a BamH1 Xho1 pgex5x - 2 T4 orfh79 pgex5x - 2 DH5α 37 PCR 1. 5 pet - 28a - orfh79 pgex5x orfh79 BL21 DE3 BL21 DE3 5 μl 5 ml Kan Amp LB ml 20 LB 37 2

3 ~ 3 h OD mol /L IPTG 1 mmol /L IPTG 1 ml Eppendoff 37 4 h 2 h 1 ml Eppendoff pet - 28a BL21 DE SDS - PAGE min 15% 20 ma 3. 5 h 1 ~ 2 h h SDS - PAGE 1. 8 Western - blot ORFH79 San Antonio Tex USA SDS - PAGE 5% PBS ph h 2 h PBS 3 10 min 1 h TBS 3 10 min NBT /BCIP 1. 9 IPTG IPTG pet - 28a - orfh79 s pet - 28a 4 LB Kan h 600 nm IPTG IPTG pgex5x orfh79 pgex - 5x - 2 LB Amp pet -28a - orfh Nco1 BamH1 BamH1 Xhol1 orfh79 pet - 28a pgex5x SDS - PAGE BL21 DE3 IPTG SDS - PAGE orfh79 pgex5x - 2 2a pet - 28a ORFH79 pgex5x orfh79 2b a b M DNA 1 pet - 28a - orfh Western blot pgex5x orfh79 Western - blot a Construction of recombinant prokaryotic expression vectors b Verifications of positive prokaryotic expression vectors M. Marker 1 Verifi- 3 IPTG pet - 28a - orfh79 3a PGEX5x orfh79 3b fication of positive pgex5x orfh79 clone by enzyme cation of positive pet - 28a - orfh79 clone by enzyme digestion 2 Veri- digestion. pgex5x orfh79 1 Western - blot Fig. 1 Construction of prokaryotic expression vector

4 3 orfh c N GST ORFH79 GST - ORFH79 a b a pgex5x orfh79 BL21 M 1 BL21 DE3 2 IPTG BL21 DE IPTG pegx5x - orfh79 BL21 DE3 4 pegx5x - orfh79 BL21 DE3 7 IPTG pgex5x - 2 BL21 DE3 8 pgex5x - 2 BL21 DE3 pgex5x orfh79 b a Expression of the pgex5x orfh79 transformant in BL21 strain M Maker 1 BL21 DE3 strain 2 TPTG induced BL21 DE3 strain IPTG induced pgex5x orfh79 transformant 7 IPTG induced empty plasmid pgex5x - 2 in BL21 DE3 strain 8 Control plasmind pgex5x - 2 in BL21 DE3 strain b Solubility analysis of the recombinant protein 1 precipitate 2 supernant. 2 Fig. 2 PGEX5x orfh79 BL21 Induced expression of fused ORFH79 protein and the solubility analysis a Western blot pet - 28a - orfh79 BL21 DE3 1 IPTG 6 h pet - 28a - orfh79 BL21 DE3 2. IPTG 6 h BL21 DE3 b Western blot pgex5x BL21 DE3 1 IPTG 6 h pgex5x orfh79 BL21 DE3 2. IPTG 6 h BL21 DE3 c Western blot pgex5x orfh79 a Western blot analysis the expression of pet - 28a - orfh79 transformant in BL21 DE3 strain. 1 pet - 28a - orfh79 transformant induced for 6 h with IPTG 2 Empty plasmid pet - 28a induced for 6 h with IPTG. b Western blot analysis the expression of pgex5x orfh79 transformant in BL21 DE3. 1 pgex5x orfh79 transformant induced for 6 h with IPTG 2 Empty plasmid pgex5x - 2 induced for 6 h with IPTG c Western blot analysis the supernant of pgex5x orfh79 recombinant after ultrasonication ORFH79 3 Fig. 3 Western blot ORFH79 Western blot assay the fused ORFH79 protein pet - 28a - orfh79 4a IPTG pet - 28a - orfh79 IPTG pet - 28a - orfh79 pet - 28a S100 pet -28a - s100

5 IPTG IPTG pet28a pgex5x orfh79 IPTG IPTG 4b N GST ORFH79 3 a pet - 28a b pgex - 5x - 2 pets100 pet - 28a - s100 peth79 pet - 28a - orfh79 5x h pgex5x orfh79 + IPTG - IPTG a Gowth curve of pet - 28a - orfh79 transformants. b Growth curve of pgex5x orfh79 transformants + Induced with IPTG - Without IPTG. 4 Fig. 4 Growth curve of orfh79 transformants 9-10 Deway T URF13 Urf13 - T 13 ku BmT - methomyl CMS T 12 Nakai 4 Duroc 6 ORF522 Orf138 5 orf79 ORF79 Nco1 PET - 28a His OrfH79 pet - 28a - orfh7 ORFH79 Western blot pet - 28a - orfh79 ORFH79 5 orf79 T ufr13 orfh79 pet - 28a - orfh79

6 3 orfh orfh79 ORFH79 ATP 8 orfh79 orfh79 orfh79 pgex ORFH79 N GST GST - ORFH79 Western - blot GST - ORFH79 OR- FH79 N GST 5 orfh79 orf79 C 5 ORF79 ORFH79 N ORFH79 1 Kaul M. Male sterility in higher plants J. Theor Appl Genet Linke B Borner T. Mitochondrial effects on flower and pollen development J. Mitochondrion Dewey R E Siedow J N Timothy D H et al. A 13 - kilodalton maize mitochondrial protein in E. coli conferssensitivity to Bipolaris maydis toxin J. Science Duroc Y Gaillard C Hiard S et al. Biochemical and functional characterization of ORF138 a mitochondrial protein responsible for Ogura cytoplasmic male sterility in Brassiceae J. Biochimie Wang Z Zou Y Li X et al. Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mrna silencing J. Plant Cell Nakai S Noda D Kondo M. High - level expression of a mitochondrial orf522 gene from the male - sterile sun ower is lethal to E. coli. Terachi J. T Breed Sci Yi P Wang L Sun Q et al. Discovery of mitochondrial chimeric gene associated with male sterility of HL - rice J. Chinese Sci Bull Peng X Wang K Hu C et al. The mitochondrial gene orfh79 plays a critical role in impairing both male gametophyte development and root growth in CMS - Honglian rice J. BMC Plant Biology Hanson M R Bentolila S. Interactions of mitochondrial and nuclear genes that affect male gametophyte development J. Plant Cell Longo E B Laun P V D Pichova A et al. Cytoplasmic male sterility A window to the world of plant mitochondrial - nuclear interactions J. Trends Genet Dewey R E Timothy D H Levings C S. Chimeric mitochondrial gens expressed in the C male - sterile cytoplasm of maize J. Curr Genet Levings C Siedow J. Molecular basis of disease susceptibility in the texas of maize J. Plant Mol Biol Wan C Li S Wen L et al. Damage of oxidative stress on mitochondria during microspores development in Honglian CMS line of rice. Plant Cell Rep J Li S Wan C Kong J. Programmed cell death during microgenesis in a Honglian CMS line of rice is correlated with oxidative stress in mitochondria J. Funct Plant Biol