- Email: [email protected]
Molecular cloning of cp gene of LSV and construction of its RNAi expression vector
Abstracts / Journal of Biotechnology 136S (2008) S183–S186
III2-P-005 NtSKP1 may affect the development of tobacco leaf Fuyun Zhang 1,∗ , Xuefang Bai 2 , Yukui Zhang 2 , Yuguang Du 2 1
Dalian Fisheries University, Dalian, PR China Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China 2
E-mail address: [email protected] (F. Zhang). The 3 end cDNA of gene SKP1 from Nicotiana tabacum var (NtSKP1) was obtained through the technique of mRNA differential display during isolated genes whose transcription were altered by oligochitosan. Because the SKP1 protein as the core component of SCF(ubiquitin ligase enzyme E3) is relevant to many plant signalling pathways, including those mediating responses to hormones, light, sucrose, developmental cues and pathogens (Ellis et al., 2002; Azevedo et al., 2002; Liu et al., 2002). To gain insight into its function, the full-length of SKP1 cDNA from Nicotiana tabacum var. samsun NN (NtSKP1) was cloned using the SMART-RACE technique, and The RNAi vector pART27-SKP1a-SKP1s was constructed. Nicotiana tabacum var. samsun NN was transformed by leaf disc method using Agrobacterium tumefaciens LBA4404 containing pART27-SKP1a-SKP1s. Whole plants were regenerated from the transformed cells. The transgene integration and transcription of putative transformants were tested by PCR. All the leaves of transformants were different from those of normal tobacco. The leaves of transgenic are crinkly similar to mosaic disease, and they change to yellow earlier compare to those of wiled type tobacco. The preliminary data suggest that NtSKP1 may play an important role in the development of tobacco leaves. Keywords: NtSKP1; RNAi; Leaf development References Azevedo, C., Sadanandom, A., Kitagawa, K., Freialdenhoven, A., Shirasu, K., SchulzeLefert, P., 2002. The RAR1 interactor SGT1, an essential component of R genetriggered disease resistance. Science 295, 2073–2076. Ellis, C., Turner, J.G., Devoto, A., 2002. Protein complexes mediate signalling in plant responses to hormones, light, sucrose and pathogens. Plant Mol. Biol. 50, 971–980. Liu, Y., Schiff, M., Marathe, R., Dinesh-Kumar, S.P., 2002. Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J. 30, 415–429.
doi:10.1016/j.jbiotec.2008.07.409 III2-P-006 Molecular cloning of cp gene of LSV and construction of its RNAi expression vector Yin Yalei ∗ , Xu Pinsan, Liu Huaxia, Li Huangai The University of Dalian Technology, Dalian, China E-mail address: [email protected] (Y. Yalei). Lily symptomless virus (LSV), a species of the genus Carlavirus, is the most prevalent virus infecting lily plants throughout the world .The CP gene of Lily symptomless viru (LSV) was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the total RNA purified from infected lily leaves. In this paper, Gateway technology is used to construct an RNAi vector containing CP gene. It is based on the site-specific recombination reaction mediated by phage . DNA fragments or targeted gene flanked by recombination sites can be transferred into vectors that contain compatible recombination sites. In LR reaction, the sense segelnents of the full
S185
length of LSV CP gene were cloned into pDONR201 Plasmid and then in BP reaction Genes in an Entry Clone can be transferred into pH7GWIWG2 Plasmid which is RNAi vector and the PCR proved that the transgene was integrated into the vector and then the RNAi vector was obtained that could be transformed into lily by Agrobac teriumtumef acien. References Bakhetia, M., Charlton, W., Atkinson, H.J., McPherson, M.J., 2005. RNA interference of dual oxidase in the plant nematode Meloidogyne incognita. Mol. Plant Microb. Interact. 18, 1099–1106. Chen, Q., Rehman, S., Smant, G., Jones, J.T., 2005. Functional analysis of pathogenicity proteins of the potato cyst nematode Globodera rostochiensis using RNAi. Mol. Plant Microb. Interact. 18, 621–625. Fire, A., Xu, S., Montgomery, M.K., et al., 1998. Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans [J]. Nature 391 (6669), 806–811. Fusaro, A.F., Matthew, L., Smith, N.A., Curtin, S.J., Dedic-Hagan, J., Ellacott, G.A., Watson, J.M., Wang, M.B., Brosnan, C., Carroll, B.J., et al., 2006. RNA interferenceinducing hairpin RNAs in plants act through the viral defence pathway. EMBO Rep. 7, 1168–1175. Helliwell, C.A., Waterhouse, P.M., 2005. Constructs and methods for hairpin RNAmediated gene silencing in plants. Methods Enzymol. 392, 24–35. Ma, Z.L., Yang, H.Y., Wang, R., et al., 2004. Construct hairpin RNA to fight against Ricedwarf virus[J]. Acta Bot. Sin. 46 (3), 332–336.
doi:10.1016/j.jbiotec.2008.07.410 III2-P-007 SNK-SPAR pathway in an induced neurotoxicity in primary cultured rat hippocampal neurons Libin Zhan 1,∗ , Xinping Niu 1 , Xiaoguang Lu 2 , Hua Sui 3 , Xiaoyang Gong 4 , Haiyan Lin 1 1 The Second Affiliated Hospital, Dalian Medical University, Dalian, China 2 Department of Emergency, Zhongshan Hospital, Dalian University, Dalian, China 3 Department of Pathophysiology, Dalian Medical University, Dalian, China 4 The First Affiliated Hospital, Dalian Medical University, Dalian, China
E-mail address: [email protected] (L. Zhan). To explore SNK-SPAR pathway in primary cultured rat hippocampal neurons and the relationship between induced neurotoxicity and SNK-SPAR pathway. The primary cultured rat hippocampal neurons were cultured; with the method of RNAi, siRNA targeted to SNK was transfected into neurons by Lipofectamine2000 for 48 h, then SNK and SPARmRNA expression were detected by RT-PCR. Neurons were exposed to 5 fibrillar 1–40, and RT-PCR was performed to detect SNK and SPARmRNA expression for different time intervals. After siRNA was transfected into neurons, SNKmRNA was downregulated, and SPARmRNA was up-regulated. With exposure to for different time intervals, we found the up-regulation of SNKmRNA expression and down-regulation of SPARmRNA, and the changes of SNK and SPARmRNA expression were most significant at 2 h. The results confirmed that SNK-SPAR pathway existed and suggested that SNK played an important role in this pathway. What’s more, could induce the changes of SNK and SPARmRNA expression and these changes had time dependent. It suggested induced neurotoxicity had close relationship with SNK-SPAR pathway. Acknowledgement This work was supported by National Natural Science Foundation of China Grant (No. 30472255).
III2-P-005 NtSKP1 may affect the development of tobacco leaf Fuyun Zhang 1,∗ , Xuefang Bai 2 , Yukui Zhang 2 , Yuguang Du 2 1
Dalian Fisheries University, Dalian, PR China Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China 2
E-mail address: [email protected] (F. Zhang). The 3 end cDNA of gene SKP1 from Nicotiana tabacum var (NtSKP1) was obtained through the technique of mRNA differential display during isolated genes whose transcription were altered by oligochitosan. Because the SKP1 protein as the core component of SCF(ubiquitin ligase enzyme E3) is relevant to many plant signalling pathways, including those mediating responses to hormones, light, sucrose, developmental cues and pathogens (Ellis et al., 2002; Azevedo et al., 2002; Liu et al., 2002). To gain insight into its function, the full-length of SKP1 cDNA from Nicotiana tabacum var. samsun NN (NtSKP1) was cloned using the SMART-RACE technique, and The RNAi vector pART27-SKP1a-SKP1s was constructed. Nicotiana tabacum var. samsun NN was transformed by leaf disc method using Agrobacterium tumefaciens LBA4404 containing pART27-SKP1a-SKP1s. Whole plants were regenerated from the transformed cells. The transgene integration and transcription of putative transformants were tested by PCR. All the leaves of transformants were different from those of normal tobacco. The leaves of transgenic are crinkly similar to mosaic disease, and they change to yellow earlier compare to those of wiled type tobacco. The preliminary data suggest that NtSKP1 may play an important role in the development of tobacco leaves. Keywords: NtSKP1; RNAi; Leaf development References Azevedo, C., Sadanandom, A., Kitagawa, K., Freialdenhoven, A., Shirasu, K., SchulzeLefert, P., 2002. The RAR1 interactor SGT1, an essential component of R genetriggered disease resistance. Science 295, 2073–2076. Ellis, C., Turner, J.G., Devoto, A., 2002. Protein complexes mediate signalling in plant responses to hormones, light, sucrose and pathogens. Plant Mol. Biol. 50, 971–980. Liu, Y., Schiff, M., Marathe, R., Dinesh-Kumar, S.P., 2002. Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J. 30, 415–429.
doi:10.1016/j.jbiotec.2008.07.409 III2-P-006 Molecular cloning of cp gene of LSV and construction of its RNAi expression vector Yin Yalei ∗ , Xu Pinsan, Liu Huaxia, Li Huangai The University of Dalian Technology, Dalian, China E-mail address: [email protected] (Y. Yalei). Lily symptomless virus (LSV), a species of the genus Carlavirus, is the most prevalent virus infecting lily plants throughout the world .The CP gene of Lily symptomless viru (LSV) was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the total RNA purified from infected lily leaves. In this paper, Gateway technology is used to construct an RNAi vector containing CP gene. It is based on the site-specific recombination reaction mediated by phage . DNA fragments or targeted gene flanked by recombination sites can be transferred into vectors that contain compatible recombination sites. In LR reaction, the sense segelnents of the full
S185
length of LSV CP gene were cloned into pDONR201 Plasmid and then in BP reaction Genes in an Entry Clone can be transferred into pH7GWIWG2 Plasmid which is RNAi vector and the PCR proved that the transgene was integrated into the vector and then the RNAi vector was obtained that could be transformed into lily by Agrobac teriumtumef acien. References Bakhetia, M., Charlton, W., Atkinson, H.J., McPherson, M.J., 2005. RNA interference of dual oxidase in the plant nematode Meloidogyne incognita. Mol. Plant Microb. Interact. 18, 1099–1106. Chen, Q., Rehman, S., Smant, G., Jones, J.T., 2005. Functional analysis of pathogenicity proteins of the potato cyst nematode Globodera rostochiensis using RNAi. Mol. Plant Microb. Interact. 18, 621–625. Fire, A., Xu, S., Montgomery, M.K., et al., 1998. Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans [J]. Nature 391 (6669), 806–811. Fusaro, A.F., Matthew, L., Smith, N.A., Curtin, S.J., Dedic-Hagan, J., Ellacott, G.A., Watson, J.M., Wang, M.B., Brosnan, C., Carroll, B.J., et al., 2006. RNA interferenceinducing hairpin RNAs in plants act through the viral defence pathway. EMBO Rep. 7, 1168–1175. Helliwell, C.A., Waterhouse, P.M., 2005. Constructs and methods for hairpin RNAmediated gene silencing in plants. Methods Enzymol. 392, 24–35. Ma, Z.L., Yang, H.Y., Wang, R., et al., 2004. Construct hairpin RNA to fight against Ricedwarf virus[J]. Acta Bot. Sin. 46 (3), 332–336.
doi:10.1016/j.jbiotec.2008.07.410 III2-P-007 SNK-SPAR pathway in an induced neurotoxicity in primary cultured rat hippocampal neurons Libin Zhan 1,∗ , Xinping Niu 1 , Xiaoguang Lu 2 , Hua Sui 3 , Xiaoyang Gong 4 , Haiyan Lin 1 1 The Second Affiliated Hospital, Dalian Medical University, Dalian, China 2 Department of Emergency, Zhongshan Hospital, Dalian University, Dalian, China 3 Department of Pathophysiology, Dalian Medical University, Dalian, China 4 The First Affiliated Hospital, Dalian Medical University, Dalian, China
E-mail address: [email protected] (L. Zhan). To explore SNK-SPAR pathway in primary cultured rat hippocampal neurons and the relationship between induced neurotoxicity and SNK-SPAR pathway. The primary cultured rat hippocampal neurons were cultured; with the method of RNAi, siRNA targeted to SNK was transfected into neurons by Lipofectamine2000 for 48 h, then SNK and SPARmRNA expression were detected by RT-PCR. Neurons were exposed to 5 fibrillar 1–40, and RT-PCR was performed to detect SNK and SPARmRNA expression for different time intervals. After siRNA was transfected into neurons, SNKmRNA was downregulated, and SPARmRNA was up-regulated. With exposure to for different time intervals, we found the up-regulation of SNKmRNA expression and down-regulation of SPARmRNA, and the changes of SNK and SPARmRNA expression were most significant at 2 h. The results confirmed that SNK-SPAR pathway existed and suggested that SNK played an important role in this pathway. What’s more, could induce the changes of SNK and SPARmRNA expression and these changes had time dependent. It suggested induced neurotoxicity had close relationship with SNK-SPAR pathway. Acknowledgement This work was supported by National Natural Science Foundation of China Grant (No. 30472255).