虾青素合成相关酶基因Adketo果实特异表达载体的构建
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摘要
目的:采用克隆的番茄E8启动子和夏侧金盏花虾青素合成相关酶基因Adketo构建果实特异表达载体,为利用植物基因工程进行虾青素生产提供新途径。方法:采用RT-PCR的方法,从夏侧金盏花中克隆到虾青素合成相关酶基因Adketo,同时以番茄基因组DNA为模板PCR扩增果实特异性启动子E8,克隆进T载体,菌液PCR初步鉴定后测序,序列分析。从T载体切下的E8启动子和Adketo基因经回收后同时插入到植物双元表达载体pBI121上,获得重组载体,然后进行菌液PCR检测和酶切鉴定。结果:测序结果分析显示获得正确的E8和Adketo基因序列。重组质粒经菌液PCR检测及酶切鉴定均获得预期大小条带。结论:该实验成功构建了番茄果实特异性E8启动子驱动虾青素合成相关酶基因Adketo的植物表达载体。
Objective:To construct the plant expression vector including Adketo gene from Adonis aestivalis and E8 promotor,and provide a new way for astaxanthin production by plant genetic engineering.Methods:The Adketo gene was clonied from Adonis aestivalis by RT-PCR and the 1.1 kb tomato E8 gene was amplified by PCR.then cloned into T vector.After the preliminary identification by Microbial PCR,the recombinant T-vectors were subjected to sequence analysis.Cut the E8 promoter and Adketo gene and inserted into the plant binary expression vector pBI121 to obtain the recombinant expression vector.And then to identify the vector by microbial PCR detection and enzyme digestion.Results:Sequencing results analysis showed the right E8 and Adketo gene sequences,the identification by microbial PCR and digestion with restriction enzymes proved that the recombinant vector had the inserts with expected length of target fragments.Conclusion:The plant expression vector containing Adketo gene driven by fruit specific promoter E8 was successfully constructed.
Objective:To construct the plant expression vector including Adketo gene from Adonis aestivalis and E8 promotor,and provide a new way for astaxanthin production by plant genetic engineering.Methods:The Adketo gene was clonied from Adonis aestivalis by RT-PCR and the 1.1 kb tomato E8 gene was amplified by PCR.then cloned into T vector.After the preliminary identification by Microbial PCR,the recombinant T-vectors were subjected to sequence analysis.Cut the E8 promoter and Adketo gene and inserted into the plant binary expression vector pBI121 to obtain the recombinant expression vector.And then to identify the vector by microbial PCR detection and enzyme digestion.Results:Sequencing results analysis showed the right E8 and Adketo gene sequences,the identification by microbial PCR and digestion with restriction enzymes proved that the recombinant vector had the inserts with expected length of target fragments.Conclusion:The plant expression vector containing Adketo gene driven by fruit specific promoter E8 was successfully constructed.
引文
1付佳,杨月欣,张立实.天然虾青素的生物保健功能及安全性概述[J].国外医学(卫生学分册),2007,34(6):382-386.
2姚康飞,张瑞莲,刘晓娟,等.不同立体构型的虾青素抗脂质过氧化活性研究[J].中国食品学报,2018,18(10):86-94.
3林晓,储小军,周蒂,等.虾青素的来源、功能及应用[J].环境与职业医学,2008,25(6):615-618.
4肖素荣,李京东.虾青素的特性及应用前景[J].中国食物与营养,2011,17(5):33-35.
5焦雪峰.虾青素在化妆品中的应用[J].广东化工,2006,1(33):382-386.
6方婷,段明慧,马晋芳,等.高压破碎提取雨生红球藻中虾青素的工艺研究[J].中药材,2018,9:1911-1914
7任伟伟,张艾青,王福强,等.海洋红酵母产虾青素发酵培养基的优化[J].粮食与饲料工业,2018,9:39-42.
8邢涛,张慧,祁琳琳,等.从雨生球藻中提取虾青素的工艺研究[J].中国食品添加剂,2018,11:169-174
9汪多仁.虾青素的开发与应用进展[J].江西食品工业,2011,3:38-41.
10 Batghare AH,Singh N,Moholkar VS.Investigations in ultrasound-induced enhancement of astaxanthin production by wild strain Phaffia rhodozyma MTCC 7536[J].Bioresource technology,2018,254:166-173.
11 Maoka T,Etoh T,Kishimoto S,et al.Carotenoids and their fatty acid esters in the petals of Adonis aestivalis[J].Journal of Oleo Science,2011,60(2):47-52.
12 Cunningham FX,Gantt E.A study in scarlet:enzymes of ketocarotenoid biosynthesis in the flowers of Adonis aestivalis[J].The Plant Journal,2005,41(3):478-492.
13高新征,李栎,邬强,等.夏侧金盏花虾青素合成相关基因Adketo和Adkc的表达[J].海南医学院学报,2012,12(18):1689-1691.
14 Ralley L,Enfissi E,Misawa N,et al.Metabolic engineering of ketocarotenoid formation in higher plants[J].The Plant Journal,2004,39(4):477-486.
15张鹏,朱宏.转基因植物生物反应器的研究[J].哈尔滨师范大学自然科学学报,2011,27(5):77-80.
16胡廷章,罗凯,甘丽萍,等.植物基因启动子的类型及其应用[J].湖北农业科学,2007,46(1):150-153.
17彭龙云.虾青素合成途径关键基因的筛选及高效细胞工厂的构建[D].广西大学,2018.
18王玉霞,李唯,王旺田,等.植物果实特异性启动子E8基因的克隆[J].华北农学报,2008,23(3):16-19.
2姚康飞,张瑞莲,刘晓娟,等.不同立体构型的虾青素抗脂质过氧化活性研究[J].中国食品学报,2018,18(10):86-94.
3林晓,储小军,周蒂,等.虾青素的来源、功能及应用[J].环境与职业医学,2008,25(6):615-618.
4肖素荣,李京东.虾青素的特性及应用前景[J].中国食物与营养,2011,17(5):33-35.
5焦雪峰.虾青素在化妆品中的应用[J].广东化工,2006,1(33):382-386.
6方婷,段明慧,马晋芳,等.高压破碎提取雨生红球藻中虾青素的工艺研究[J].中药材,2018,9:1911-1914
7任伟伟,张艾青,王福强,等.海洋红酵母产虾青素发酵培养基的优化[J].粮食与饲料工业,2018,9:39-42.
8邢涛,张慧,祁琳琳,等.从雨生球藻中提取虾青素的工艺研究[J].中国食品添加剂,2018,11:169-174
9汪多仁.虾青素的开发与应用进展[J].江西食品工业,2011,3:38-41.
10 Batghare AH,Singh N,Moholkar VS.Investigations in ultrasound-induced enhancement of astaxanthin production by wild strain Phaffia rhodozyma MTCC 7536[J].Bioresource technology,2018,254:166-173.
11 Maoka T,Etoh T,Kishimoto S,et al.Carotenoids and their fatty acid esters in the petals of Adonis aestivalis[J].Journal of Oleo Science,2011,60(2):47-52.
12 Cunningham FX,Gantt E.A study in scarlet:enzymes of ketocarotenoid biosynthesis in the flowers of Adonis aestivalis[J].The Plant Journal,2005,41(3):478-492.
13高新征,李栎,邬强,等.夏侧金盏花虾青素合成相关基因Adketo和Adkc的表达[J].海南医学院学报,2012,12(18):1689-1691.
14 Ralley L,Enfissi E,Misawa N,et al.Metabolic engineering of ketocarotenoid formation in higher plants[J].The Plant Journal,2004,39(4):477-486.
15张鹏,朱宏.转基因植物生物反应器的研究[J].哈尔滨师范大学自然科学学报,2011,27(5):77-80.
16胡廷章,罗凯,甘丽萍,等.植物基因启动子的类型及其应用[J].湖北农业科学,2007,46(1):150-153.
17彭龙云.虾青素合成途径关键基因的筛选及高效细胞工厂的构建[D].广西大学,2018.
18王玉霞,李唯,王旺田,等.植物果实特异性启动子E8基因的克隆[J].华北农学报,2008,23(3):16-19.