| 桑树1-氨基环丙烷-1-羧酸氧化酶基因(MnACO)启动子功能分析 |
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| 引用本文： | 余建,刘长英,赵爱春,王传宏,蔡雨翔,余茂德.桑树1-氨基环丙烷-1-羧酸氧化酶基因(MnACO)启动子功能分析[J].作物学报,2017,43(6):839-848. |
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| 作者姓名： | 余建 刘长英 赵爱春 王传宏 蔡雨翔 余茂德 |
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| 作者单位： | 西南大学生物技术学院 / 西南大学家蚕基因组生物学国家重点实验室，重庆 400715 |
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| 基金项目： | 本研究由国家公益性行业(农业)科研专项经费项目(201403064)，重庆市研究生科研创新项目(CYS2015070)，国家现代农业产业技术体系建设专项(CARS-22)资助。 |
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| 摘 要： | 1-氨基环丙烷-1-羧酸氧化酶(ACO)作为关键酶,能够催化1-氨基环丙烷-1-羧酸(ACC)形成乙烯。为探究桑树MnACO基因在桑树生长发育和抵御外界胁迫中的功能,本研究构建了p MnACO::GUS的植物表达载体并转化拟南芥。采用GUS组织染色法鉴定转基因拟南芥不同生长阶段及胁迫处理后的GUS活性。通过PCR克隆得到MnACO1和MnACO2启动子片段,它们分别为1518 bp和1429 bp,启动子区域有大量的TATA-box、CAAT-box和其他响应外界刺激的顺式作用元件。GUS活性分析显示MnACO启动子能驱动GUS在拟南芥中表达;MnACO1启动子能驱动GUS在拟南芥的根、叶片、花瓣、花药、花丝、柱头以及果荚中表达,且活性较MnACO2强;MnACO2启动子不能驱动GUS在果荚中无表达。转MnACO1和MnACO2植株经不同逆境处理后GUS表达活性不同,转MnACO1植株的GUS活性随处理延长时间而减弱,转MnACO2植株GUS活性随处理时间延长而增强。q RT-PCR检测2周苗龄的桑幼苗在经过胁迫处理后Ma ACO1和Ma ACO2的基因表达量,发现Ma ACO基因的表达模式与MnACO启动子GUS活性变化趋势一致。本研究结果表明,MnACO为诱导型启动子,MnACO1兼具组成型启动子特性,MnACO2兼具组织特异型启动子特性。MnACO1在转基因植株中对胁迫响应能力更强,预示着可将其用来调控改良桑树品种抗逆性靶基因;Ma ACO2可能与果实成熟有关,可将其启动子作为果实特异性启动子对桑椹品质进行合理改良。
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| 关 键 词： | 桑树 乙烯 ACO 启动子 GUS 功能分析 |
| 收稿时间： | 2016-07-25 |
Functional Analysis of 1-Aminocyclopropane-1-carboxylate Oxidase Gene’s Promoter in Mulberry |
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| YU Jian,LIU Chang-Ying,ZHAO Ai-Chun,WANG Chuan-Hong,CAI Yu-Xiang,YU Mao-De.Functional Analysis of 1-Aminocyclopropane-1-carboxylate Oxidase Gene’s Promoter in Mulberry[J].Acta Agronomica Sinica,2017,43(6):839-848. |
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| Authors: | YU Jian LIU Chang-Ying ZHAO Ai-Chun WANG Chuan-Hong CAI Yu-Xiang YU Mao-De |
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| Institution: | College of Biotechnology, Southwest University / State Key Laboratory of Silkworm Genome Biology, Chongqing 400715, China? |
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| Abstract: | 1-Aminocyclopropane-1-carboxylate oxidase (ACO) as a key enzyme catalyzes the reaction from 1-Aminocyclopropane- 1-carboxylate (ACC) to ethylene. In order to explore the function of MnACO in mulberry growth and development and resisting external stresses, we constructed pMnACO::GUS fusion and transformed it into Arabidopsis thaliana, and used GUS histochemical staining to identify GUS activities of transgenic Arabidopsis thaliana treated by different stresses in different growth stages . MnACO1 and MnACO2 promoter fragments obtained by PCR were 1518 bp and 1429 bp, respectively. There were lots of cis-acting elements such as TATA-box, CAAT-box and others responded to external stimuli. MnACO promoter could drive GUS to express in Arabidopsis thaliana; MnACO1 promoter expressed in the root, leaf, flower petal, anther, filament, stigma and silique of Arabidopsis thaliana and their activities were higher than those of MnACO2; but there was no expression of MnACO2 promoter in silique. The pMnACO1::GUS and pMnACO2::GUS transgenetic plants had different activities in different treatments. The GUS activities of the pMnACO1::GUS transgenetic plants were weakened, while those of the pMnACO2::GUS transgenetic plants were strengthened with the elongation of stress treatment time. Detection of MaACO expression in stress treatment of 2-week-old seedlings by qRT-PCRindicated that the trends of expression pattern of MaACO and its GUS activity changes were almost the same. This research indicated that MnACO is inducible promoter, MnACO1 has the characteristic of constitutive promoter, and MnACO2 has the characteristic of tissue-specific promoter. MnACO1 had more ability to respond to stresses in the transgenic plant, indicating it can be used to regulate the target gene of improving mulberry stress resistance. MaACO2 might be relevant to fruit maturation, thus its promoter could be used as fruit-specific promoter to improve the quality of mulberry fruit. |
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| Keywords: | Mulberry Ethylene ACO Promoter GUS Functional Analysis |
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