ZHANG Yu,YUAN Jinhua,TANG Minghao,et al.Cloning and Expression Analysis of Flavanone-3-Hydroxylase Gene and Promoter From Vitis amurensis[J].Northern Horticulture,2025,(12):1-7.[doi:10.11937/bfyy.20244333]
山葡萄黄烷酮-3-羟化酶基因及其启动子的克隆与表达分析
- Title:
- Cloning and Expression Analysis of Flavanone-3-Hydroxylase Gene and Promoter From Vitis amurensis
- 文章编号:
- 1001-0009(2025)12-0001-07
- Keywords:
- Vitis amurensis; flavanone-3-hydroxylase; promoter; cis-acting element
- 分类号:
- S 663
- 文献标志码:
- A
- 摘要:
- 以山葡萄（Vitis amurensis）‘双红’品种为试材，采用CTAB法提取山葡萄果皮中RNA与叶片中cDNA，对‘双红’黄烷酮-3-羟化酶基因(flavanone-3-hydroxylase，F3H）及其启动子进行克隆，并进行F3H蛋白质的原核表达和启动子不同缺失片段的瞬时表达,研究了山葡萄不同种类花色苷的形成机理，以期为花色苷的实际生产提供参考依据。结果表明：‘双红’品种F3H完整开放阅读框1 091 bp，启动子序列1 454 bp，采用生物信息学软件分析发现，F3H理化性质较 ‘双丰’品种有所差异，其启动子序列中除具有核心启动子元件CAAT-box和TATA-box外，还有一系列光响应元件，以及其他参与不同种类生物调控的顺式作用元件，转化烟草叶片进行瞬时表达后，β-葡萄糖苷酸酶（GUS）活性有所差异，随着启动子缺失片段长度的增加而呈现先下降后上升的趋势。通过对F3H重组蛋白体外表达条件的探究，当控制变量温度为25 ℃、培养时间为7 h、IPTG浓度为0.8 mmol·L-1的条件下，上清样品中目的蛋白表达量最高。
- Abstract:
- Taking the Vitis amurensis‘Shuanghong’ variety as the test material，RNA was extracted from the skin and cDNA was extracted from the leaves of Vitis amurensis used CTAB method.The flavanone-3-hydroxylase gene (F3H) and its promoter were cloned，and the prokaryotic expression of F3H protein and transient expression of different deleted fragments of the promoter were performed.The formation mechanism of different types of anthocyanins in Vitis amurensis were studied in order to provide reference for the actual production of anthocyanins.The results showed that the complete open reading frame of the F3H gene was cloned to 1 091 bp and the promoter sequence was 1 454 bp.Bioinformatics software analysis revealed that the physicochemical properties of F3H in Vitis amurensis were different from those in ‘Shuangfeng’.In addition to the core promoter elements CAAT-box and TATA-box，there were a series of light-responsive elements and cis involved in different types of biological regulation in the promoter sequence.After transient expression in tobacco leaves，the GUS activity showed differences and showed a trend of first decreasing and then increasing with the length of the missing promoter fragments.By exploring the in vitro expression conditions of F3H recombinant protein，it was found that the highest expression level of the target protein was observed in the supernatant sample under controlled variable temperature of 25 ℃，culture time of 7 hours，and IPTG concentration of 0.8 mmol·L-1.
参考文献/References:
［1］曹芳芳,李娟,刘海峰.山葡萄DFR基因全长cDNA的克隆与序列分析［J］.江苏农业科学,2016,44(7):34-38.［2］刘薇,王阿娜,万德光,等.桑叶f3h基因PCR克隆与分析［J］.时珍国医国药,2017,28(3):756-757.［3］张琼,王红清,冷平,等.草莓果实发育过程中花色苷和黄酮醇类物质的形成机制［J］.园艺学报,2008,35(12):1735-1741.［4］赵权,王军,韩富亮.不同品种山葡萄酒中花色苷的成分分析［J］.西北农林科技大学学报(自然科学版),2013,41(6):195-201.［5］郑晓瑜.辽宁碱蓬BADH启动子盐诱导功能元件鉴定及转录因子分离［D］.大连:辽宁师范大学,2011.［6］吴文华,李新舟.水稻中受硝酸盐诱导的基因启动子的克隆与植物双元表达载体的构建［J］.湖北大学学报(自然科学版),2007,29(3):294-297.［7］蒋淑丽,李涛.植物启动子及相关功能研究概述［J］.陕西农业科学,2012,58(5):94-97.［8］王薇.抗逆相关转录因子基因GmDREB3启动子分析及兼抗白粉病、条锈病小麦分子标记检测［D］.乌鲁木齐:新疆农业大学,2009.［9］张录华.番茄SlTFT3基因的启动子分析［J］.南方农业,2018,12(18):176-177.［10］郝迪萩,赵琳,陈李淼,等.克隆启动子方法的比较及应用［J］.东北农业大学学报,2010,41(3):154-160.［11］王军,贺普超.山葡萄基因组DNA提取及RAPD鉴定［J］.果树科学,2000,17(2):79-82.［12］李旭娟,林秀琴,字秋艳,等.甘蔗ScMOC1基因启动子的克隆与瞬时表达分析［J］.植物遗传资源学报,2019,20(3):709-717.［13］FORKMANN G,HELLER W,GRISEBACH H.Anthocyanin biosynthesis in flowers of Matthiola incana flavanone 3-and flavonoid 3′-hydroxylases［J］.Zeitschrift Für Naturforschung C,1980,35(9/10):691-695.［14］肖啸,王旺田,孙萍,等.不同葡萄品种CBF1基因的克隆及序列分析［J］.生物技术通报,2011,27(12):82-87.［15］文铁桥.基因工程原理［M］.北京:科学出版社,2014.［16］张磊,唐永凯,李红霞,等.促进原核表达蛋白可溶性的研究进展［J］.中国生物工程杂志,2021,41(S1):138-149.［17］张宇,刘海峰.山葡萄F3′5′H基因的克隆与表达及其启动子5′端缺失片段的功能分析［J］.扬州大学学报(农业与生命科学版),2022,43(3):26-33.［18］张宇,徐智慧,任邵琦,等.山葡萄F3′H基因及其启动子的克隆与表达分析［J］.农业生物技术学报,2021,29(11):2099-2108.［19］ZHAO P X，MIAO Z Q，ZHANG J，et al.Arabidopsis MADS-box factor AGL16 negatively regulates drought resistance via stomatal density and stomatal movement［J］.Journal of Experimental Botany，2020，71(19)：6092-6106.［20］MATUS J T,LOYOLA R,VEGA A,et al.Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera［J］.Journal of Experimental Botany,2009,60(3):853-867.［21］陈建琦,赵明珠,王义,等.植物中组织特异性启动子的研究进展［J］.北方园艺，2023(19)：128-134.
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备注/Memo
第一作者简介：张宇（1995-），女，硕士，助理研究员，现主要从事山葡萄的基因克隆等研究工作。E-mail：1142157796@qq.com.责任作者：刘海峰（1989-），男，博士，副教授，硕士生导师，现主要从事山葡萄基因克隆等研究工作。E-mail：Liufenge_1989@163.com.基金项目：吉林省农业科学院创新工程资助项目（KYJF2024SJ011）。收稿日期：2024-11-04