[1]张倩,孙晶,廖静,等.RNA干扰技术抑制洋桔梗ACC合酶基因的表达[J].江苏农业学报,2017,(02):267-272.[doi:doi:10.3969/j.issn.1000-4440.2017.02.005]
　ZHANG Qian,SUN Jing,LIAO Jing,et al.Inhibition of ACC synthase gene expression in Eustoma grandiflorum using RNA interference technology[J].,2017,(02):267-272.[doi:doi:10.3969/j.issn.1000-4440.2017.02.005]
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RNA干扰技术抑制洋桔梗ACC合酶基因的表达()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2017年02期
页码:
267-272
栏目:
遗传育种·生理生化
出版日期:
2017-04-30

文章信息/Info

Title:
Inhibition of ACC synthase gene expression in Eustoma grandiflorum using RNA interference technology
作者:
张倩; 孙晶; 廖静; 陈崇顺
(南京师范大学生命科学学院/江苏省生物多样性与生物技术重点实验室,江苏南京210023)
Author(s):
ZHANG Qian; SUN Jing; LIAO Jing; CHEN Chong-shun
(College of Life Sciences, Nanjing Normal University / Jiangsu Key Laboratory of Biodiversity and Biotechnology, Nanjing 210023, China)
关键词:
洋桔梗; ACC合酶; RNA干扰; 花期
Keywords:
Eustoma grandiflorum; ACC synthase; RNA interference; flowering time
分类号:
S681.9
DOI:
doi:10.3969/j.issn.1000-4440.2017.02.005
文献标志码:
A
摘要:
为了延长洋桔梗的花期，本研究通过农杆菌介导法，以ACC合酶（ACS）基因干扰表达载体，对Double Mariachi Pink洋桔梗（Eustoma grandiflorum）的叶片外植体进行了转化。经过再生芽的筛选增殖培养，在含有卡那霉素Km（15 mg/L）再生根筛选培养基中得到了46株抗性苗，其中11株经GFP基因PCR鉴定为阳性。经进一步的GFP基因RT-PCR鉴定，3株表达为阳性，并且其中2株经GFP荧光检测呈阳性。提取荧光阳性植株RNA，逆转录后，进行了ACS荧光定量PCR。结果表明，与未转基因对照植株相比，转基因植株ACS的相对表达量平均下降了63.17%。花期统计结果表明，ACS干扰表达载体转化的洋桔梗植株花期比未转基因植株对照延长了17 d。
Abstract:
To prolong the life of cut flower of Eustoma grandiflorum, leaf explants of Double Mariachi Pink were transformed with the ACC synthase (ACS) gene interference expression vector by Agrobacterium tumefaciens-mediated method. Forty six resistant regenerated plantlets rooted in the culture medium containing kanamycin (15 mg/L), 11 of which were identified as positive by PCR of GFP gene. RT-PCR revealed that, three plantlets were positive for expression, and two of them were positive by GFP fluorescence detection. The ACS fluorescence quantitative PCR was performed after the fluorescence-positive plant RNA was extracted and the RNA was reversely transcribed. The results showed that the ACS relative expression in the transformed lines was decreased by an average of 63.17%, compared with the untransformed control. The flowering time for the transformed plant was also markedly prolonged (17 days longer).

参考文献/References:

［1]BRENT H. Flower breeding and genetics[M]. Germany: Springer Netherlands, 2006: 644-663.
［2]罗江会，马婧，刘道凤，等. 乙烯对蜡梅切花开放衰老及乙烯受体基因表达的影响[J]. 植物生理学报，2015, 51 (2): 253-258.
［3]ICHIMURA K, SHIMAMURA M, HISAMATSU T. Role of ethylene in senescence of cut Eustoma flowers [J]. Postharvest Biol Tec, 1998, 14(2):193-198.
［4]田如英，周恒. 不同保鲜剂对洋桔梗鲜切花保鲜效果的影响[J].植物生理学报，2005, 41(5)：625-626.
［5]DEIKMAN J. Molecular mechanism of ethylene regulation of gene transcription [J]. Physiologia Plantarum, 1997, 100(3):561-566.
［6]KOSUGI Y, MATSUOKA A, HIGASHI A,et al. 2-Aminooxyisobutyric acid inhibits the in vitro activities of both 1-Aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase in ethylene biosynthetic pathway and prolongs vase life of cut carnation flowers[J]. Journal of Plant Biology, 2014, 57(4): 218-224.
［7]NAKAJIMA N, ITOH T, TAKIKAWA S, et al. Improvement in ozone tolerance of tobacco plants with an antisense DNA for 1‐aminocyclopropane-1-carboxylate synthase[J]. Plant, Cell & Environment, 2002, 25(6): 727-735.
［8]陈银华，张俊红，欧阳波. 花椰菜ACC氧化酶基因的克隆及其RNAi对内源基因表达的抑制作用[J]. 遗传学报, 2005, 32(7):764-769.
［9]周雅智，姜玲，黄勇. BraSDG2RNA干扰载体的构建和拟南芥的遗传转化［J］．　山东农业科学，2016,48(10）:7-11.
［10]周晓婴,付三雄,陈松,等. 甘蓝型油菜CRABS CLAW 基因克隆及其RNA 干扰载体的构建[J]. 江苏农业学报,2015,31(4):737-742.
［11]MATZKE M, MATZKE A J M, KOOTER J M. RNA: guiding gene silencing[J]. Science, 2001, 293(5532): 1080-1083.
［12]杜改梅，胡志华，罗碧平，等. RNA干扰沉默Ghrelin基因对仔猪胃酸分泌的调节作用[J]. 南方农业学报，2016，47(8)：1396-1400.
［13]廖静. 洋桔梗ACC合酶基因的RNA干扰研究[D]. 南京：南京师范大学, 2011：5.
［14]王关林，方洪筠. 植物基因工程原理与技术[M]. 北京：科学出版社,1998.
［15]杨燕燕，陈崇顺，瞿大枞，等. 洋桔梗高频再生系统的建立及其卡那霉素敏感性测定[J]. 江苏农业科学, 2007(2):98-100.
［16]廖静，王轶，陈崇顺，等. 洋桔梗Double Mariachi Pink高效遗传转化体系的建立[J]. 江苏农业科学, 2011, 39(2):91-94.
［17]楼程富，陆小平，潘刚.桑树遗传转化的受体细胞及其再生[J]. 蚕业科学, 2005, 31(2):182-186.
［18]马燕斌，孙璇，吴霞，等. 转新型抗草甘膦基因棉花的转化效率及相关分子特征分析[J].棉花学报, 2016, 28(4): 418-424.
［19]盖树鹏，孟祥栋. 转基因植物的筛选与检测[J]. 山东农业大学学报(自然科学版), 2000, 31(1):95-100.
［20]HONG B, MA C, YANG Y, et al. Over-expression of AtDREB1A in chrysanthemum enhances tolerance to heat stress [J]. Plant Molecular Biology, 2009, 70(3):231-240.
［21]PEEBLES C A M, HUGHES E H, SHANKS J V, et al. Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time[J]. Metabolic Engineering, 2009, 11(2): 76-86.
［22]AGINDOTAN B O, SHIEL P J, BERGER P H. Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan real-time RT-PCR[J]. Journal of Virological Methods, 2007, 142(1): 1-9.
［23]HANNON G J. RNA interference [J]. Nature, 2002, 418(6894):244-251.
［24]TANG G, GALILI G. Using RNAi to improve plant nutritional value: from mechanism to application [J]. Trends in Biotechnology, 2004, 22(9):463-469.
［25]LIU Q, SINGH S P, GREEN A G. High-stearic and high-oleic cottonseed oils produced by hairbin RNA-mediated post-transcriptional gene silencing [J]. Plant Physiology, 2002, 129(4):1732-1743.
［26]孙晶，王轶，陈崇顺，等. ACC合酶反义基因转化洋桔梗Double Mariachi Pink的研究[J].江苏农业科学，2012, 40(4):27-31. ［1]BRENT H. Flower breeding and genetics[M]. Germany: Springer Netherlands, 2006: 644-663.
［2]罗江会，马婧，刘道凤，等. 乙烯对蜡梅切花开放衰老及乙烯受体基因表达的影响[J]. 植物生理学报，2015, 51 (2): 253-258.
［3]ICHIMURA K, SHIMAMURA M, HISAMATSU T. Role of ethylene in senescence of cut Eustoma flowers [J]. Postharvest Biol Tec, 1998, 14(2):193-198.
［4]田如英，周恒. 不同保鲜剂对洋桔梗鲜切花保鲜效果的影响[J].植物生理学报，2005, 41(5)：625-626.
［5]DEIKMAN J. Molecular mechanism of ethylene regulation of gene transcription [J]. Physiologia Plantarum, 1997, 100(3):561-566.
［6]KOSUGI Y, MATSUOKA A, HIGASHI A,et al. 2-Aminooxyisobutyric acid inhibits the in vitro activities of both 1-Aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase in ethylene biosynthetic pathway and prolongs vase life of cut carnation flowers[J]. Journal of Plant Biology, 2014, 57(4): 218-224.
［7]NAKAJIMA N, ITOH T, TAKIKAWA S, et al. Improvement in ozone tolerance of tobacco plants with an antisense DNA for 1‐aminocyclopropane-1-carboxylate synthase[J]. Plant, Cell & Environment, 2002, 25(6): 727-735.
［8]陈银华，张俊红，欧阳波. 花椰菜ACC氧化酶基因的克隆及其RNAi对内源基因表达的抑制作用[J]. 遗传学报, 2005, 32(7):764-769.
［9]周雅智，姜玲，黄勇. BraSDG2RNA干扰载体的构建和拟南芥的遗传转化［J］．　山东农业科学，2016,48(10）:7-11.
［10]周晓婴,付三雄,陈松,等. 甘蓝型油菜CRABS CLAW 基因克隆及其RNA 干扰载体的构建[J]. 江苏农业学报,2015,31(4):737-742.
［11]MATZKE M, MATZKE A J M, KOOTER J M. RNA: guiding gene silencing[J]. Science, 2001, 293(5532): 1080-1083.
［12]杜改梅，胡志华，罗碧平，等. RNA干扰沉默Ghrelin基因对仔猪胃酸分泌的调节作用[J]. 南方农业学报，2016，47(8)：1396-1400.
［13]廖静. 洋桔梗ACC合酶基因的RNA干扰研究[D]. 南京：南京师范大学, 2011：5.
［14]王关林，方洪筠. 植物基因工程原理与技术[M]. 北京：科学出版社,1998.
［15]杨燕燕，陈崇顺，瞿大枞，等. 洋桔梗高频再生系统的建立及其卡那霉素敏感性测定[J]. 江苏农业科学, 2007(2):98-100.
［16]廖静，王轶，陈崇顺，等. 洋桔梗Double Mariachi Pink高效遗传转化体系的建立[J]. 江苏农业科学, 2011, 39(2):91-94.
［17]楼程富，陆小平，潘刚.桑树遗传转化的受体细胞及其再生[J]. 蚕业科学, 2005, 31(2):182-186.
［18]马燕斌，孙璇，吴霞，等. 转新型抗草甘膦基因棉花的转化效率及相关分子特征分析[J].棉花学报, 2016, 28(4): 418-424.
［19]盖树鹏，孟祥栋. 转基因植物的筛选与检测[J]. 山东农业大学学报(自然科学版), 2000, 31(1):95-100.
［20]HONG B, MA C, YANG Y, et al. Over-expression of AtDREB1A in chrysanthemum enhances tolerance to heat stress [J]. Plant Molecular Biology, 2009, 70(3):231-240.
［21]PEEBLES C A M, HUGHES E H, SHANKS J V, et al. Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time[J]. Metabolic Engineering, 2009, 11(2): 76-86.
［22]AGINDOTAN B O, SHIEL P J, BERGER P H. Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan real-time RT-PCR[J]. Journal of Virological Methods, 2007, 142(1): 1-9.
［23]HANNON G J. RNA interference [J]. Nature, 2002, 418(6894):244-251.
［24]TANG G, GALILI G. Using RNAi to improve plant nutritional value: from mechanism to application [J]. Trends in Biotechnology, 2004, 22(9):463-469.
［25]LIU Q, SINGH S P, GREEN A G. High-stearic and high-oleic cottonseed oils produced by hairbin RNA-mediated post-transcriptional gene silencing [J]. Plant Physiology, 2002, 129(4):1732-1743.
［26]孙晶，王轶，陈崇顺，等. ACC合酶反义基因转化洋桔梗Double Mariachi Pink的研究[J].江苏农业科学，2012, 40(4):27-31. ［1]BRENT H. Flower breeding and genetics[M]. Germany: Springer Netherlands, 2006: 644-663.
［2]罗江会，马婧，刘道凤，等. 乙烯对蜡梅切花开放衰老及乙烯受体基因表达的影响[J]. 植物生理学报，2015, 51 (2): 253-258.
［3]ICHIMURA K, SHIMAMURA M, HISAMATSU T. Role of ethylene in senescence of cut Eustoma flowers [J]. Postharvest Biol Tec, 1998, 14(2):193-198.
［4]田如英，周恒. 不同保鲜剂对洋桔梗鲜切花保鲜效果的影响[J].植物生理学报，2005, 41(5)：625-626.
［5]DEIKMAN J. Molecular mechanism of ethylene regulation of gene transcription [J]. Physiologia Plantarum, 1997, 100(3):561-566.
［6]KOSUGI Y, MATSUOKA A, HIGASHI A,et al. 2-Aminooxyisobutyric acid inhibits the in vitro activities of both 1-Aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase in ethylene biosynthetic pathway and prolongs vase life of cut carnation flowers[J]. Journal of Plant Biology, 2014, 57(4): 218-224.
［7]NAKAJIMA N, ITOH T, TAKIKAWA S, et al. Improvement in ozone tolerance of tobacco plants with an antisense DNA for 1‐aminocyclopropane-1-carboxylate synthase[J]. Plant, Cell & Environment, 2002, 25(6): 727-735.
［8]陈银华，张俊红，欧阳波. 花椰菜ACC氧化酶基因的克隆及其RNAi对内源基因表达的抑制作用[J]. 遗传学报, 2005, 32(7):764-769.
［9]周雅智，姜玲，黄勇. BraSDG2RNA干扰载体的构建和拟南芥的遗传转化［J］．　山东农业科学，2016,48(10）:7-11.
［10]周晓婴,付三雄,陈松,等. 甘蓝型油菜CRABS CLAW 基因克隆及其RNA 干扰载体的构建[J]. 江苏农业学报,2015,31(4):737-742.
［11]MATZKE M, MATZKE A J M, KOOTER J M. RNA: guiding gene silencing[J]. Science, 2001, 293(5532): 1080-1083.
［12]杜改梅，胡志华，罗碧平，等. RNA干扰沉默Ghrelin基因对仔猪胃酸分泌的调节作用[J]. 南方农业学报，2016，47(8)：1396-1400.
［13]廖静. 洋桔梗ACC合酶基因的RNA干扰研究[D]. 南京：南京师范大学, 2011：5.
［14]王关林，方洪筠. 植物基因工程原理与技术[M]. 北京：科学出版社,1998.
［15]杨燕燕，陈崇顺，瞿大枞，等. 洋桔梗高频再生系统的建立及其卡那霉素敏感性测定[J]. 江苏农业科学, 2007(2):98-100.
［16]廖静，王轶，陈崇顺，等. 洋桔梗Double Mariachi Pink高效遗传转化体系的建立[J]. 江苏农业科学, 2011, 39(2):91-94.
［17]楼程富，陆小平，潘刚.桑树遗传转化的受体细胞及其再生[J]. 蚕业科学, 2005, 31(2):182-186.
［18]马燕斌，孙璇，吴霞，等. 转新型抗草甘膦基因棉花的转化效率及相关分子特征分析[J].棉花学报, 2016, 28(4): 418-424.
［19]盖树鹏，孟祥栋. 转基因植物的筛选与检测[J]. 山东农业大学学报(自然科学版), 2000, 31(1):95-100.
［20]HONG B, MA C, YANG Y, et al. Over-expression of AtDREB1A in chrysanthemum enhances tolerance to heat stress [J]. Plant Molecular Biology, 2009, 70(3):231-240.
［21]PEEBLES C A M, HUGHES E H, SHANKS J V, et al. Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time[J]. Metabolic Engineering, 2009, 11(2): 76-86.
［22]AGINDOTAN B O, SHIEL P J, BERGER P H. Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan real-time RT-PCR[J]. Journal of Virological Methods, 2007, 142(1): 1-9.
［23]HANNON G J. RNA interference [J]. Nature, 2002, 418(6894):244-251.
［24]TANG G, GALILI G. Using RNAi to improve plant nutritional value: from mechanism to application [J]. Trends in Biotechnology, 2004, 22(9):463-469.
［25]LIU Q, SINGH S P, GREEN A G. High-stearic and high-oleic cottonseed oils produced by hairbin RNA-mediated post-transcriptional gene silencing [J]. Plant Physiology, 2002, 129(4):1732-1743.
［26]孙晶，王轶，陈崇顺，等. ACC合酶反义基因转化洋桔梗Double Mariachi Pink的研究[J].江苏农业科学，2012, 40(4):27-31. ［1]BRENT H. Flower breeding and genetics[M]. Germany: Springer Netherlands, 2006: 644-663.
［2]罗江会，马婧，刘道凤，等. 乙烯对蜡梅切花开放衰老及乙烯受体基因表达的影响[J]. 植物生理学报，2015, 51 (2): 253-258.
［3]ICHIMURA K, SHIMAMURA M, HISAMATSU T. Role of ethylene in senescence of cut Eustoma flowers [J]. Postharvest Biol Tec, 1998, 14(2):193-198.
［4]田如英，周恒. 不同保鲜剂对洋桔梗鲜切花保鲜效果的影响[J].植物生理学报，2005, 41(5)：625-626.
［5]DEIKMAN J. Molecular mechanism of ethylene regulation of gene transcription [J]. Physiologia Plantarum, 1997, 100(3):561-566.
［6]KOSUGI Y, MATSUOKA A, HIGASHI A,et al. 2-Aminooxyisobutyric acid inhibits the in vitro activities of both 1-Aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase in ethylene biosynthetic pathway and prolongs vase life of cut carnation flowers[J]. Journal of Plant Biology, 2014, 57(4): 218-224.
［7]NAKAJIMA N, ITOH T, TAKIKAWA S, et al. Improvement in ozone tolerance of tobacco plants with an antisense DNA for 1‐aminocyclopropane-1-carboxylate synthase[J]. Plant, Cell & Environment, 2002, 25(6): 727-735.
［8]陈银华，张俊红，欧阳波. 花椰菜ACC氧化酶基因的克隆及其RNAi对内源基因表达的抑制作用[J]. 遗传学报, 2005, 32(7):764-769.
［9]周雅智，姜玲，黄勇. BraSDG2RNA干扰载体的构建和拟南芥的遗传转化［J］．　山东农业科学，2016,48(10）:7-11.
［10]周晓婴,付三雄,陈松,等. 甘蓝型油菜CRABS CLAW 基因克隆及其RNA 干扰载体的构建[J]. 江苏农业学报,2015,31(4):737-742.
［11]MATZKE M, MATZKE A J M, KOOTER J M. RNA: guiding gene silencing[J]. Science, 2001, 293(5532): 1080-1083.
［12]杜改梅，胡志华，罗碧平，等. RNA干扰沉默Ghrelin基因对仔猪胃酸分泌的调节作用[J]. 南方农业学报，2016，47(8)：1396-1400.
［13]廖静. 洋桔梗ACC合酶基因的RNA干扰研究[D]. 南京：南京师范大学, 2011：5.
［14]王关林，方洪筠. 植物基因工程原理与技术[M]. 北京：科学出版社,1998.
［15]杨燕燕，陈崇顺，瞿大枞，等. 洋桔梗高频再生系统的建立及其卡那霉素敏感性测定[J]. 江苏农业科学, 2007(2):98-100.
［16]廖静，王轶，陈崇顺，等. 洋桔梗Double Mariachi Pink高效遗传转化体系的建立[J]. 江苏农业科学, 2011, 39(2):91-94.
［17]楼程富，陆小平，潘刚.桑树遗传转化的受体细胞及其再生[J]. 蚕业科学, 2005, 31(2):182-186.
［18]马燕斌，孙璇，吴霞，等. 转新型抗草甘膦基因棉花的转化效率及相关分子特征分析[J].棉花学报, 2016, 28(4): 418-424.
［19]盖树鹏，孟祥栋. 转基因植物的筛选与检测[J]. 山东农业大学学报(自然科学版), 2000, 31(1):95-100.
［20]HONG B, MA C, YANG Y, et al. Over-expression of AtDREB1A in chrysanthemum enhances tolerance to heat stress [J]. Plant Molecular Biology, 2009, 70(3):231-240.
［21]PEEBLES C A M, HUGHES E H, SHANKS J V, et al. Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time[J]. Metabolic Engineering, 2009, 11(2): 76-86.
［22]AGINDOTAN B O, SHIEL P J, BERGER P H. Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan real-time RT-PCR[J]. Journal of Virological Methods, 2007, 142(1): 1-9.
［23]HANNON G J. RNA interference [J]. Nature, 2002, 418(6894):244-251.
［24]TANG G, GALILI G. Using RNAi to improve plant nutritional value: from mechanism to application [J]. Trends in Biotechnology, 2004, 22(9):463-469.
［25]LIU Q, SINGH S P, GREEN A G. High-stearic and high-oleic cottonseed oils produced by hairbin RNA-mediated post-transcriptional gene silencing [J]. Plant Physiology, 2002, 129(4):1732-1743.
［26]孙晶，王轶，陈崇顺，等. ACC合酶反义基因转化洋桔梗Double Mariachi Pink的研究[J].江苏农业科学，2012, 40(4):27-31.

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备注/Memo

备注/Memo:
收稿日期：2016-07-31 基金项目：江苏省自然科学基金重点项目（BK2001206） 作者简介：张倩（1994-），女，安徽蚌埠人，硕士研究生，主要从事植物生物技术与分子生物学研究。（Tel）15996270610；(E-mail) QianZhangNNU@163. com。孙 晶为共同第一作者。 通讯作者：陈崇顺，（Tel）13815862346；(E-mail)chenchongshun@njnu.edu.cn
更新日期/Last Update: 2017-05-02