水稻GluB-4终止子的克隆与功能分析
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摘要
目前转基因常用的终止子大多为细菌或真菌来源,有可能引起转基因食品安全性问题。本研究以水稻(Oryzasativa)‘kitaake’为材料,克隆了谷蛋白B-4基因(GluB-4)的终止子GluB-4-T(GenBank登记号:X14393),分析了其在不同启动子驱动下替代根癌农杆菌(Agrobacterium tumefaciens)胭脂碱合酶终止子(nos-T)的可能性。β-d-葡糖醛酸酶(GUS)组织化学染色结果表明, GluB-4-T在GluB-3启动子驱动下,未改变GluB-3启动子的组织表达特异性,但GUS表达强度发生了改变; GUS活性分析显示GluB-4-T GUS活性是nos-T的2.4倍。为验证GluB-4-T增强外源基因表达的特性不依赖于上游启动子序列,将其克隆到强的胚乳特异性启动子GluC和组成型启动子OsActin下, GUS活性分别是nos-T的2.0和2.2倍,说明GluB-4-T增强外源基因表达的特性不依赖于上游启动子序列。以上结果表明GluB-4-T在水稻体内发挥正常的转录终止活性,且比nos-T表达活性强,可以作为有效终止子用于遗传转化,避免多基因转化系统中由于转基因同源性过高引起的转基因沉默现象。该结果为实现外源目的基因在水稻胚乳中特异表达提供理论依据。
The bacterial-or fungal-origion terminator is commonly used in transgenic research, which raises great concerns about the safety of genetically modified foods. In this study, we cloned glutelin B-4 terminator(Glu B-4-T, GenBank number X14393) and evaluated the potential that Glu B-4-T function as terminators in enhancing the expression of the GUS reporter gene driven by glutelin GluB-3, GluC and OsActin promoters in stable transgenic rice(Oryza sativa) lines. The results show that Glu B-4-T significantly enhanced the activity of Glu B-3 promoter without changing its tissue specificity but altering its expression intensity in endosperm. With GluB-4-T, the expression of GUS was higher by 2.4 fold than with the nos terminator(nos-T). GluB-4-T combined with Glu C and Os Actin promoters, also increased GUS levels by 2.0 and 2.2 folds, respectively, in stable transgenic rice line, which indicates that the enhancements were independent of the promoter sequences. The results suggest that GluB-4-T has normal transcriptional termination activity in rice and is more potent than nos-T. Glu B-4-T might be an ideal candidate for genetic transformation and it could function as faithful terminator in mono-or multi-gene transformation avoiding homology-based gene silencing. The results above lay foundation for the specific expression in rice endosperm.
The bacterial-or fungal-origion terminator is commonly used in transgenic research, which raises great concerns about the safety of genetically modified foods. In this study, we cloned glutelin B-4 terminator(Glu B-4-T, GenBank number X14393) and evaluated the potential that Glu B-4-T function as terminators in enhancing the expression of the GUS reporter gene driven by glutelin GluB-3, GluC and OsActin promoters in stable transgenic rice(Oryza sativa) lines. The results show that Glu B-4-T significantly enhanced the activity of Glu B-3 promoter without changing its tissue specificity but altering its expression intensity in endosperm. With GluB-4-T, the expression of GUS was higher by 2.4 fold than with the nos terminator(nos-T). GluB-4-T combined with Glu C and Os Actin promoters, also increased GUS levels by 2.0 and 2.2 folds, respectively, in stable transgenic rice line, which indicates that the enhancements were independent of the promoter sequences. The results suggest that GluB-4-T has normal transcriptional termination activity in rice and is more potent than nos-T. Glu B-4-T might be an ideal candidate for genetic transformation and it could function as faithful terminator in mono-or multi-gene transformation avoiding homology-based gene silencing. The results above lay foundation for the specific expression in rice endosperm.
引文
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Buntru M,G?rtner S,Staib L,et al(2013).Delivery of multiple transgenes to plant cells by an improved version of MultiRound Gateway technology.Transgenic Res,22:153-167
Cai C,Zhuang Y,Zhang C,et al(2014).Cloning and functional analysis of terminator in tobacco root-specific NtR19gene.Mol Plant Breeding,12(3):466-471(in Chinese with English abstract)[蔡翠雅,庄宇慧,张冲等(2014).烟草根特异NtR19基因终止子的克隆及功能分析.分子植物育种,12(3):466-471]
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Chen Z,Wang GJ,An XM(2013).Cloning and transient expression analysis of PtrAP1-2 promoter from Populus trichocarpa Torr.&Gray.Plant Physiol J,49(6):591-597(in Chinese with English abstract)[陈仲,王佳,安新民(2013).毛果杨PtrAP1-2基因启动子的克隆及其瞬时表达分析.植物生理学报,49(6):591-597]
de Moor CH,Meijer H,Lissenden S(2005).Mechanisms of translational control by the 3′UTR in development and differentiation.Semin Cell Dev Biol,16(1):49-58
Depicker A,Stachel S,Dhaese P,et al(1982).Nopaline synthase:transcript mapping and DNA sequence.J Mol Appl Genet,1:561-573
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Jefferson RA,Kavanagh TA,Bevan MW(1987).GUS fusions:β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO J,6:3901-3907
Jiao GL,Meng ZH,Nie AQ,et al(2004).Expression of CaMV 35S promoter in transgenic cotton.Acta Agron Sin,30(11):1135-1139(in Chinese with English abstract)[焦改丽,孟钊红,聂安全等(2004).花椰菜花叶病毒(CaMV)35S启动子在转基因棉花中的表达.作物学报,30(11):1135-1139
Jin SX,Han J,Liu XY,et al(2007).Chloroplast DNA extraction in cotton by SDS-proteinase method and cloning of the promoter and terminator of psbA gene.Mol Plant Breed,5(5):683-689(in Chinese with English abstract)[金双侠,韩杰,刘小云等(2007).SDS-蛋白酶法分离棉花cpDNA及psbA基因启动子、终止子克隆.分子植物育种,5(5):683-689]
Li J,Qu SH(2012).PolyA trapping by using terminatorless RFP reporter gene in rice.Acta Agr Zhejiangensis,24:758-763(in Chinese with English abstract)[李军,瞿绍洪(2012).用无终止子的RFP报告基因捕获水稻终止子研究.浙江农业学报,24:758-763]
Li WJ,Dai LL,Chai ZJ,et al(2012).Evaluation of seed storage protein gene 3′-untranslated regions in enhancing gene expression in transgenic rice seed.Transgenic Res,21:545-553
Niu DD,Hao YJ,Rong RJ,et al(2014).Detection of GUSprotein and its expression pattern in transgenic rice plants.Sci Agr Sin,47(14):2715-2722(in Chinese with English abstract)[牛东东,郝育杰,荣瑞娟等(2014).转基因水稻中GUS蛋白质的检测及其表达特征.中国农业科学,47(14):2715-2722]
Qu LQ,Takaiwa F(2004).Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice.Plant Biotechnol J,2:113-125
Qu LQ,Xing YP,Liu WX,et al(2008).Expression pattern and activity of six glutelin gene promoters in transgenic rice.J Exp Bot,59(9):2417-2424
Shou HX,Zhou L(2017).Genetic engineering and genome editing in plants.Plant Physiology J,53(8):1341-1344(in Chinese with English abstract)[寿惠霞,周丽(2017).植物转基因与基因组编辑.植物生理学报,53(8):1341-1344]
Wang XT,Huang S,Xu ZS,et al(2017).Application of Pisum sativum terminator rbc-T in wheat transformation.Acta Agron Sin,43(8):1254-1258(in Chinese with English abstract)[王小婷,黄锁,徐兆师等(2017).豌豆终止子rbc-T在转基因小麦研究中的应用,作物学报,43(8):1254-1258]
Yang L,Wakasa Y,Kawakatsu T,et al(2009).The 3′-untranslated region of rice glutelin Glu B-1 affects accumulation of heterologous protein in transgenic rice.Biotechnol Lett,31:1625-1631
Yuan S,Huang R,Luo H(2008).GUS reporter gene for analysis of gene function in plants.Acta Agron Sin,22(5):310-314(in Chinese)[袁思玮,黄锐之,罗红兵(2008).GUS报告基因在植物功能基因研究中的应用.作物学报,22(5):310-314]
Zhang FY,Yin WB,Hu ZM(2007).Cloning and function identification of the promoters and terminator of Populus tomentosa chloroplast genes.J Agr Biotechnol,15(3):439-445[张发云,尹维波,胡赞民(2007).毛白杨叶绿体基因启动子和终止子的克隆及其功能鉴定.农业生物技术学报,15(3):439-445]
Zhang R,Guo S(2001).Study on plant insect-resistant genetic engineering.Biotechnol Bull,(2):8-12[张锐,郭三堆(2001).植物抗虫基因工程研究进展.生物技术通报,(2):8-12]
Buntru M,G?rtner S,Staib L,et al(2013).Delivery of multiple transgenes to plant cells by an improved version of MultiRound Gateway technology.Transgenic Res,22:153-167
Cai C,Zhuang Y,Zhang C,et al(2014).Cloning and functional analysis of terminator in tobacco root-specific NtR19gene.Mol Plant Breeding,12(3):466-471(in Chinese with English abstract)[蔡翠雅,庄宇慧,张冲等(2014).烟草根特异NtR19基因终止子的克隆及功能分析.分子植物育种,12(3):466-471]
Chen QJ,Zhou HM,Chen J,et al(2006).A Gateway-based platform for multigene plant transformation.Plant Mol Biol,62(6):927-936
Chen Z,Wang GJ,An XM(2013).Cloning and transient expression analysis of PtrAP1-2 promoter from Populus trichocarpa Torr.&Gray.Plant Physiol J,49(6):591-597(in Chinese with English abstract)[陈仲,王佳,安新民(2013).毛果杨PtrAP1-2基因启动子的克隆及其瞬时表达分析.植物生理学报,49(6):591-597]
de Moor CH,Meijer H,Lissenden S(2005).Mechanisms of translational control by the 3′UTR in development and differentiation.Semin Cell Dev Biol,16(1):49-58
Depicker A,Stachel S,Dhaese P,et al(1982).Nopaline synthase:transcript mapping and DNA sequence.J Mol Appl Genet,1:561-573
Hiei Y,Ohta S,Komari T,et al(1994).Efficient transformation of rice(Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant J,6(2):271-282
Jefferson RA,Kavanagh TA,Bevan MW(1987).GUS fusions:β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO J,6:3901-3907
Jiao GL,Meng ZH,Nie AQ,et al(2004).Expression of CaMV 35S promoter in transgenic cotton.Acta Agron Sin,30(11):1135-1139(in Chinese with English abstract)[焦改丽,孟钊红,聂安全等(2004).花椰菜花叶病毒(CaMV)35S启动子在转基因棉花中的表达.作物学报,30(11):1135-1139
Jin SX,Han J,Liu XY,et al(2007).Chloroplast DNA extraction in cotton by SDS-proteinase method and cloning of the promoter and terminator of psbA gene.Mol Plant Breed,5(5):683-689(in Chinese with English abstract)[金双侠,韩杰,刘小云等(2007).SDS-蛋白酶法分离棉花cpDNA及psbA基因启动子、终止子克隆.分子植物育种,5(5):683-689]
Li J,Qu SH(2012).PolyA trapping by using terminatorless RFP reporter gene in rice.Acta Agr Zhejiangensis,24:758-763(in Chinese with English abstract)[李军,瞿绍洪(2012).用无终止子的RFP报告基因捕获水稻终止子研究.浙江农业学报,24:758-763]
Li WJ,Dai LL,Chai ZJ,et al(2012).Evaluation of seed storage protein gene 3′-untranslated regions in enhancing gene expression in transgenic rice seed.Transgenic Res,21:545-553
Niu DD,Hao YJ,Rong RJ,et al(2014).Detection of GUSprotein and its expression pattern in transgenic rice plants.Sci Agr Sin,47(14):2715-2722(in Chinese with English abstract)[牛东东,郝育杰,荣瑞娟等(2014).转基因水稻中GUS蛋白质的检测及其表达特征.中国农业科学,47(14):2715-2722]
Qu LQ,Takaiwa F(2004).Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice.Plant Biotechnol J,2:113-125
Qu LQ,Xing YP,Liu WX,et al(2008).Expression pattern and activity of six glutelin gene promoters in transgenic rice.J Exp Bot,59(9):2417-2424
Shou HX,Zhou L(2017).Genetic engineering and genome editing in plants.Plant Physiology J,53(8):1341-1344(in Chinese with English abstract)[寿惠霞,周丽(2017).植物转基因与基因组编辑.植物生理学报,53(8):1341-1344]
Wang XT,Huang S,Xu ZS,et al(2017).Application of Pisum sativum terminator rbc-T in wheat transformation.Acta Agron Sin,43(8):1254-1258(in Chinese with English abstract)[王小婷,黄锁,徐兆师等(2017).豌豆终止子rbc-T在转基因小麦研究中的应用,作物学报,43(8):1254-1258]
Yang L,Wakasa Y,Kawakatsu T,et al(2009).The 3′-untranslated region of rice glutelin Glu B-1 affects accumulation of heterologous protein in transgenic rice.Biotechnol Lett,31:1625-1631
Yuan S,Huang R,Luo H(2008).GUS reporter gene for analysis of gene function in plants.Acta Agron Sin,22(5):310-314(in Chinese)[袁思玮,黄锐之,罗红兵(2008).GUS报告基因在植物功能基因研究中的应用.作物学报,22(5):310-314]
Zhang FY,Yin WB,Hu ZM(2007).Cloning and function identification of the promoters and terminator of Populus tomentosa chloroplast genes.J Agr Biotechnol,15(3):439-445[张发云,尹维波,胡赞民(2007).毛白杨叶绿体基因启动子和终止子的克隆及其功能鉴定.农业生物技术学报,15(3):439-445]
Zhang R,Guo S(2001).Study on plant insect-resistant genetic engineering.Biotechnol Bull,(2):8-12[张锐,郭三堆(2001).植物抗虫基因工程研究进展.生物技术通报,(2):8-12]