- Email: [email protected]
Identification and cloning of up-regulated proteins in posterior silk glands of Antheraea pernyi larvae in fifth instar
Abstracts / Journal of Biotechnology 136S (2008) S217–S231
IV1-P-007 Identification and cloning of up-regulated proteins in posterior silk glands of Antheraea pernyi larvae in fifth instar Shurong Xu ∗ , Wenli Li School of Environmental & Biological Science & Technology, Dalian University of Technology, Dalian, China E-mail address: xu [email protected] (S. Xu). Antheraea pernyi is a typical wild silkworm which silk gland is an organ specialized for the synthesis and secretion of silk proteins. This study was focused on the protein changes of silk glands during fifth instar to help elucidate the mechanism of metabolism. Two-dimensional gel electrophoresis was performed on posterior silk glands of fifth instar (Wu et al., 2007; Zhang et al., 2006). Through silver staining, about 700 spots arranging the pH of 4–7 were resolved on each gel. Comparing the profile of day 1 and day 4 with imagemaster, 10 up-regulated proteins were excised from the gel and detected by MAIDI-TOF/TOF. Seven of which were identified by searching against the protein sequences from the NCBInr databases, using the MASCOT search program. They are proteins related to DNA replication, transcription and translation and silk secretion. The other three were not identified because of the insufficient database. Of the identified spots, glutathione S-transferase theta (GSTT) and ribosomal protein L8 (RPL8) were studied ulteriorly. According to the sequences of silkworm and other insects of Lepidaptera, complete length of the DNA sequences of these two proteins were cloned through degenerate primer reverse transcriptase-polymerase chain reaction and deposited in GenBank (Yamamoto et al., 2005). The accession numbers are EU541490 and EU541491. GSTT shows 89.35% homology with B. mori, while RPL8 shares 100%. Also real-time PCR revealed that the two genes expression level of day 4 was both obviously higher than which of the former days. References Wu, W.C., Gao, Q.K., Chen, J.E., Qian, Y.W., Li, J.Y., Lu, H.Y., Meng, Z.Q., Ni, C.X., Zhong, B., 2007. Analysis of two-dimensional gel electrophoresis images of protein from posterior silk gland of silkworm (Bombyx mori) on day 1 and day 4 in the 5th instar stage. Agric. Sci. China Sinica 6, 249–254. Yamamoto, K., Zhang, P., Miake, F., Kashige, N., Aso, Y., Banno, Y., Fujii, H., 2005. Cloning, expression and characterization of the theta-class glutathione Stransferase from the silkworm the silkworm, Bommbyx mori. Comp. Biochem. Phys. part B Japan 141, 340–346. Zhang, P., Aso, Y., Yamamoto, K., Banno, Y., Wang, Y.Q., Tsuchida, K., Kawaguchi, Y., Fujii, 2006. Proteome analysis of silk gland proteins from the silkworm, Bombyx mori. Protemics, Japan 6, 2586–2599.
S221
receptors, respectively. In mammals and plants, PTS1 and PTS2 import both require PEX5 (Lee et al., 2006; Braverman et al., 1998); while in yeast, the presents of Pex20p allow the Pex5p and Pex7p function independently (Purdue et al., 1998). In filamentous fungi, Pex20p was also found in several genomes, whereas the direct data still lack on whether PEX5 interact with PEX7 and thus affect the PTS2 import. To answer these questions, we investigated the PTS1 and PTS2 localization in Magnaporthe grisea, a well-known model fungus. First, by ATMT mediated gene disruption, the PEX5 and PEX7 orthologues (namely MGPEX5 and MGPEX7 here) were disrupted in M. grisea Guy11 strain. Then, GFP fusion with PTS1 (SKL) and PTS2 (N-terminal of thiolase of Saccharomyces cerevisiae) were constructed and introduced into wild type, mgpex5 and mgpex7, respectively. Under a laser confocal microscope, green fluorescence was observed in a punctuated pattern which emerged more frequently at the periphery of the conidia in the Guy11/GPFPTS1 and Guy11/GFP-PTS2 strains, consistent with the peroxisomal localization. While in the mgpex5/GFP-PTS1 and mgpex7/GFPPTS2 strains, fluorescence was cytoplasmic, indicating the protein import was blocked by MGPEX5 or MGPEX7 mutation. In the mgpex5/GFP-PTS1 and mgpex7/GFP-PTS2 strains, fluorescence was still punctuated, similarly as the wild type strain. Taking together, we conclude that the PTS1 and PTS2 receptor function independently in M. grisea; i.e., MGPEX5 serves only for PTS1 import and MGPEX7 only for PTS2. Acknowledgement This work was supported by grant from Zhejiang Natural Science Foundation (No. Y306638). References Braverman, N., Dodt, G., Gould, S.J., Valle, D., 1998. An isoform of Pex5p, the human PTS1 receptor, is required for the import of PTS2 proteins into peroxisomes. Hum. Mol. Genetics 7, 1195–1205. Lee, J., Jang, H., Park, J., Jung, J., Lee, S., Park, S., Chi, Y., Moon, J., Lee, Y., Kim, S., Kim, J., Yun, D., Cho, M., Lee, K., Lee, S., 2006. Cloning of two splice variants of the rice PTS1 receptor, OsPex5pL and OsPex5pS, and their functional characterization using pex5-deficient yeast and Arabidopsis. Plant J. 47, 457–466. Purdue, P.E., Yang, X., Lazarow, P.B., 1998. Pex18p and Pex21p, a novel pair of related peroxins essential for peroxisomal targeting by the PTS2 pathway. J. Cell Biol. 143, 1859–1869.
doi:10.1016/j.jbiotec.2008.07.467 IV1-P-009
doi:10.1016/j.jbiotec.2008.07.466
GUS mediated dual screen and a M-PCR method in targeted gene disruption in Magnaporthe grisea
IV1-P-008
Jiaoyu Wang ∗ , Zhen Zhang, Xinfa Du, Rongyao Chai, Haiping Qiu, Guochang Sun
Fluorescence localization of PTS1 and PTS2 in PEX5 and PEX7 mutants of Magnaporthe grisea
Zhejiang Academy of Agricultural Science, Hangzhou 310021, China
Jiaoyu Wang 1,∗ , Xiaoyan Wu 2 , Zhen Zhang, Xinfa Du 1 , Xueqin Mao 1 , Guochang Sun 1 1
Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Science, Hangzhou 310021, China 2 College of Plant Protection, Northwest Agriculture and Forest University, Yangling 712100, China E-mail addresses: [email protected] (J. Wang), [email protected] (G. Sun). Transport of peroxisomal matrix proteins depends on two types of targeting signals: PTS1 and PTS2. PEX5 and PEX7 encode their
E-mail addresses: [email protected] [email protected] (G. Sun).
(J.
Wang),
The positive–negative dual screen (DS) has been already applied to targeted gene disruption in several fungal species to enrich the targeted events (Takahashi et al., 2004; Gardiner and Howlett, 2004). The -1, 3-glucanase/GUS was often used as a marker in plants (Miao and Lam, 1995), whereas still unreported in DS of fungi. To establish a GUS mediated DS system (GUS-DS), we first checked the endogenous GUS activity in different fungal species by X-Gluc assay. Seventy-eight strains in more than 20 species were tested, most of which were GUS− with three exceptions. Further, the gusA transformants of Magnaporthe grisea show high GUS activity. So,
IV1-P-007 Identification and cloning of up-regulated proteins in posterior silk glands of Antheraea pernyi larvae in fifth instar Shurong Xu ∗ , Wenli Li School of Environmental & Biological Science & Technology, Dalian University of Technology, Dalian, China E-mail address: xu [email protected] (S. Xu). Antheraea pernyi is a typical wild silkworm which silk gland is an organ specialized for the synthesis and secretion of silk proteins. This study was focused on the protein changes of silk glands during fifth instar to help elucidate the mechanism of metabolism. Two-dimensional gel electrophoresis was performed on posterior silk glands of fifth instar (Wu et al., 2007; Zhang et al., 2006). Through silver staining, about 700 spots arranging the pH of 4–7 were resolved on each gel. Comparing the profile of day 1 and day 4 with imagemaster, 10 up-regulated proteins were excised from the gel and detected by MAIDI-TOF/TOF. Seven of which were identified by searching against the protein sequences from the NCBInr databases, using the MASCOT search program. They are proteins related to DNA replication, transcription and translation and silk secretion. The other three were not identified because of the insufficient database. Of the identified spots, glutathione S-transferase theta (GSTT) and ribosomal protein L8 (RPL8) were studied ulteriorly. According to the sequences of silkworm and other insects of Lepidaptera, complete length of the DNA sequences of these two proteins were cloned through degenerate primer reverse transcriptase-polymerase chain reaction and deposited in GenBank (Yamamoto et al., 2005). The accession numbers are EU541490 and EU541491. GSTT shows 89.35% homology with B. mori, while RPL8 shares 100%. Also real-time PCR revealed that the two genes expression level of day 4 was both obviously higher than which of the former days. References Wu, W.C., Gao, Q.K., Chen, J.E., Qian, Y.W., Li, J.Y., Lu, H.Y., Meng, Z.Q., Ni, C.X., Zhong, B., 2007. Analysis of two-dimensional gel electrophoresis images of protein from posterior silk gland of silkworm (Bombyx mori) on day 1 and day 4 in the 5th instar stage. Agric. Sci. China Sinica 6, 249–254. Yamamoto, K., Zhang, P., Miake, F., Kashige, N., Aso, Y., Banno, Y., Fujii, H., 2005. Cloning, expression and characterization of the theta-class glutathione Stransferase from the silkworm the silkworm, Bommbyx mori. Comp. Biochem. Phys. part B Japan 141, 340–346. Zhang, P., Aso, Y., Yamamoto, K., Banno, Y., Wang, Y.Q., Tsuchida, K., Kawaguchi, Y., Fujii, 2006. Proteome analysis of silk gland proteins from the silkworm, Bombyx mori. Protemics, Japan 6, 2586–2599.
S221
receptors, respectively. In mammals and plants, PTS1 and PTS2 import both require PEX5 (Lee et al., 2006; Braverman et al., 1998); while in yeast, the presents of Pex20p allow the Pex5p and Pex7p function independently (Purdue et al., 1998). In filamentous fungi, Pex20p was also found in several genomes, whereas the direct data still lack on whether PEX5 interact with PEX7 and thus affect the PTS2 import. To answer these questions, we investigated the PTS1 and PTS2 localization in Magnaporthe grisea, a well-known model fungus. First, by ATMT mediated gene disruption, the PEX5 and PEX7 orthologues (namely MGPEX5 and MGPEX7 here) were disrupted in M. grisea Guy11 strain. Then, GFP fusion with PTS1 (SKL) and PTS2 (N-terminal of thiolase of Saccharomyces cerevisiae) were constructed and introduced into wild type, mgpex5 and mgpex7, respectively. Under a laser confocal microscope, green fluorescence was observed in a punctuated pattern which emerged more frequently at the periphery of the conidia in the Guy11/GPFPTS1 and Guy11/GFP-PTS2 strains, consistent with the peroxisomal localization. While in the mgpex5/GFP-PTS1 and mgpex7/GFPPTS2 strains, fluorescence was cytoplasmic, indicating the protein import was blocked by MGPEX5 or MGPEX7 mutation. In the mgpex5/GFP-PTS1 and mgpex7/GFP-PTS2 strains, fluorescence was still punctuated, similarly as the wild type strain. Taking together, we conclude that the PTS1 and PTS2 receptor function independently in M. grisea; i.e., MGPEX5 serves only for PTS1 import and MGPEX7 only for PTS2. Acknowledgement This work was supported by grant from Zhejiang Natural Science Foundation (No. Y306638). References Braverman, N., Dodt, G., Gould, S.J., Valle, D., 1998. An isoform of Pex5p, the human PTS1 receptor, is required for the import of PTS2 proteins into peroxisomes. Hum. Mol. Genetics 7, 1195–1205. Lee, J., Jang, H., Park, J., Jung, J., Lee, S., Park, S., Chi, Y., Moon, J., Lee, Y., Kim, S., Kim, J., Yun, D., Cho, M., Lee, K., Lee, S., 2006. Cloning of two splice variants of the rice PTS1 receptor, OsPex5pL and OsPex5pS, and their functional characterization using pex5-deficient yeast and Arabidopsis. Plant J. 47, 457–466. Purdue, P.E., Yang, X., Lazarow, P.B., 1998. Pex18p and Pex21p, a novel pair of related peroxins essential for peroxisomal targeting by the PTS2 pathway. J. Cell Biol. 143, 1859–1869.
doi:10.1016/j.jbiotec.2008.07.467 IV1-P-009
doi:10.1016/j.jbiotec.2008.07.466
GUS mediated dual screen and a M-PCR method in targeted gene disruption in Magnaporthe grisea
IV1-P-008
Jiaoyu Wang ∗ , Zhen Zhang, Xinfa Du, Rongyao Chai, Haiping Qiu, Guochang Sun
Fluorescence localization of PTS1 and PTS2 in PEX5 and PEX7 mutants of Magnaporthe grisea
Zhejiang Academy of Agricultural Science, Hangzhou 310021, China
Jiaoyu Wang 1,∗ , Xiaoyan Wu 2 , Zhen Zhang, Xinfa Du 1 , Xueqin Mao 1 , Guochang Sun 1 1
Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Science, Hangzhou 310021, China 2 College of Plant Protection, Northwest Agriculture and Forest University, Yangling 712100, China E-mail addresses: [email protected] (J. Wang), [email protected] (G. Sun). Transport of peroxisomal matrix proteins depends on two types of targeting signals: PTS1 and PTS2. PEX5 and PEX7 encode their
E-mail addresses: [email protected] [email protected] (G. Sun).
(J.
Wang),
The positive–negative dual screen (DS) has been already applied to targeted gene disruption in several fungal species to enrich the targeted events (Takahashi et al., 2004; Gardiner and Howlett, 2004). The -1, 3-glucanase/GUS was often used as a marker in plants (Miao and Lam, 1995), whereas still unreported in DS of fungi. To establish a GUS mediated DS system (GUS-DS), we first checked the endogenous GUS activity in different fungal species by X-Gluc assay. Seventy-eight strains in more than 20 species were tested, most of which were GUS− with three exceptions. Further, the gusA transformants of Magnaporthe grisea show high GUS activity. So,