叶用莴苣无缝克隆构建LsE3基因过表达载体和新RNAi载体及遗传转化体系优化

doi:10.11924/j.issn.1000-6850.casb2020-0345

中国农学通报 ›› 2021, Vol. 37 ›› Issue (11): 15-23.doi: 10.11924/j.issn.1000-6850.casb2020-0345

所属专题：生物技术

叶用莴苣无缝克隆构建LsE3基因过表达载体和新RNAi载体及遗传转化体系优化

王婷甄(), 孙燕川, 唐文琨, 范双喜(), 郝敬虹()

北京农学院植物科学技术学院/农业应用新技术北京市重点实验室,北京 102206

收稿日期:2020-08-10 修回日期:2020-11-16 出版日期:2021-04-15 发布日期:2021-04-13
通讯作者: 范双喜,郝敬虹
作者简介:王婷甄,女,1995年出生,浙江东阳人,硕士研究生,研究方向：叶用莴苣生理和分子机制。通信地址：102206 北京市昌平区回龙观镇北农路7号北京农学院科技综合楼A区404室,E-mail： 1398932310@qq.com。
基金资助:
国家自然科学基金面上项目“促分裂原活化蛋白激酶MAPK4调控叶用莴苣高温抽薹的作用机制研究”(31972400);2018年北京市自然科学基金-北京市教委联合重点项目“生长素响应因子ARF调控也用莴苣高温抽薹的作用机制”(KZ201810020027);现代农业产业技术体系北京市叶类蔬菜创新团队“生菜育种”(BAIC07-2021);2020年北京农学院学位与研究生教育改革与发展项目“北京金惠农农业专业合作社实践基地建设”;高精尖学科建设项目“蔬菜优质高效生产”

Construction of LsE3 Gene Overexpression Vector and a New RNAi Vector by Seamless Cloning and Optimization of Genetic Transformation System of Leaf Lettuce

Wang Tingzhen(), Sun Yanchuan, Tang Wenkun, Fan Shuangxi(), Hao Jinghong()

College of Plant Science and Technology, Beijing University of Agriculture/Beijing Key Laboratory for Agricultural Application and New Technique, Beijing 102206

Received:2020-08-10 Revised:2020-11-16 Online:2021-04-15 Published:2021-04-13
Contact: Fan Shuangxi,Hao Jinghong

摘要/Abstract

摘要：

研究旨在应用新技术和新载体构建过表达和沉默载体,优化遗传转化体系,验证生菜LsE3基因功能,探究高温抽薹机制。以pCAMBIA1304和pFGC5941载体为材料,通过无缝克隆构建叶用莴苣转基因载体,并通过筛选培养基配方优化遗传转化体系。试验成功地以pCAMBIA1304为基础,插入源自pFGC5941的dsRNA表达框序列,获得了新RNAi空载pCAMBIA1304- FGC5941,构建了LsE3沉默载体,并成功以双元质粒载体pCAMBIA1304构建了LsE3过表达载体。试验对易抽薹叶用莴苣品种‘GB-31’进行潮霉素浓度、直接芽诱导培养基配方和生根培养基配方的筛选,确立了潮霉素适宜筛选浓度为5 mg/L,并确立了最优培养芽诱导和生根培养基配方。试验成功获得新RNAi空载并构建了转基因载体,优化了遗传转化体系,为解决载体构建繁琐复杂的问题提供新思路,并奠定了生菜转基因技术的基础。

关键词: 叶用莴苣, 高温抽薹, LsE3泛素连接酶, 沉默载体, 载体构建, 过表达, RNAi, 遗传转化

Abstract:

The aims of this study are to construct overexpression and silence vectors by using new technologies and new vectors, optimize the genetic transformation system, verify the function of LsE3 gene and explore the mechanism of high temperature bolting in lettuce. Using pCAMBIA1304 and pFGC5941 as materials, the transgenic vector of lettuce was constructed by seamless cloning, and the genetic transformation system was optimized by screening medium formula. Based on pCAMBIA1304, the dsRNA expression frame sequence derived from pFGC5941 was successfully inserted into pCAMBIA1304. The new RNAi empty vector pCAMBIA1304-pFGC5941 was obtained, LsE3 silencing vector was constructed. And LsE3 overexpression vector was successfully constructed by binary plasmid vector pCAMBIA1304. Hygromycin concentration, direct bud induction medium formula and rooting medium formula of easy bolting leaf lettuce variety ‘GB-31’ were screened in this experiment. The suitable concentration of hygromycin was determined to be 5 mg/L, and the optimal medium formula for bud induction and rooting was established. This experiment successfully obtained a new RNAi empty vector and transgenic vectors, and optimized the genetic transformation system, which provided a new idea for solving the complex problem of vector construction, and laid the foundation of lettuce transgenic technology.

Key words: leaf lettuce, high temperature bolting, ubiquitin ligase LsE3, silence vectors, vector construction, overexpression, RNAi, genetic transformation

中图分类号:

S636.2

王婷甄, 孙燕川, 唐文琨, 范双喜, 郝敬虹. 叶用莴苣无缝克隆构建LsE3基因过表达载体和新RNAi载体及遗传转化体系优化[J]. 中国农学通报, 2021, 37(11): 15-23.

Wang Tingzhen, Sun Yanchuan, Tang Wenkun, Fan Shuangxi, Hao Jinghong. Construction of LsE3 Gene Overexpression Vector and a New RNAi Vector by Seamless Cloning and Optimization of Genetic Transformation System of Leaf Lettuce[J]. Chinese Agricultural Science Bulletin, 2021, 37(11): 15-23.

图/表 10

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引物名称	引物序列(5’-3’)
F	cgggggactcttgacATGTTGGCTCCAAGTCCATCTG
R	gaaattcgagctggtcaccCTAGACCTCCTCACAAGCACTG
iF	cgggggactcttgacCGCTCGAGTATAAGAGCTCT
iR	gaaattcgagctggtcaccGGATCCAAATACCTGCAAATTG
Sense-F	tacatttacaattaccatggGCAAAGCGACTGGGCTTATTC
Sense-R	tcatcgattgggcgcgccCTAATGTAACATACTGGCTGCC
Antisense-F	tttggatccggtgaccCTAATGTAACATACTGGCTGCC
Antisense-R	attcgagctggtcaccGCAAAGCGACTGGGCTTATTC
Lac Z	GCAGCTTGAGCTTGGATCAG
NOS	GATAATCATCGCAAGACCGG

引物名称	引物序列(5’-3’)
F	cgggggactcttgacATGTTGGCTCCAAGTCCATCTG
R	gaaattcgagctggtcaccCTAGACCTCCTCACAAGCACTG
iF	cgggggactcttgacCGCTCGAGTATAAGAGCTCT
iR	gaaattcgagctggtcaccGGATCCAAATACCTGCAAATTG
Sense-F	tacatttacaattaccatggGCAAAGCGACTGGGCTTATTC
Sense-R	tcatcgattgggcgcgccCTAATGTAACATACTGGCTGCC
Antisense-F	tttggatccggtgaccCTAATGTAACATACTGGCTGCC
Antisense-R	attcgagctggtcaccGCAAAGCGACTGGGCTTATTC
Lac Z	GCAGCTTGAGCTTGGATCAG
NOS	GATAATCATCGCAAGACCGG

培养基编号	NAA/(mg/L)	6-BA/(mg/L)	KT/(mg/L)	活性炭/(g/L)
M1^[14]	0.05	0.1	-	-
M2^[15]	0.1	0.2	-	-
M3^[16]	0.1	0.2	0.2	-
M4^[17]	0.5	10	-	0.2

培养基编号	NAA/(mg/L)	6-BA/(mg/L)	KT/(mg/L)	活性炭/(g/L)
M1^[14]	0.05	0.1	-	-
M2^[15]	0.1	0.2	-	-
M3^[16]	0.1	0.2	0.2	-
M4^[17]	0.5	10	-	0.2

培养基编号	NAA/(mg/L)	IBA/(mg/L)	MS盐/(g/L)	蔗糖/(g/L)	琼脂/(g/L)	活性炭/(g/L)
R1^[18]	-	-	2.2	30	7	-
R2^[14]	0.03	-	2.2	30	7	-
R3^[19,20]	0.5	-	2.2	30	7	0.5
R4^[17]	-	1	4.4	30	7	-

叶用莴苣无缝克隆构建LsE3基因过表达载体和新RNAi载体及遗传转化体系优化

Construction of LsE3 Gene Overexpression Vector and a New RNAi Vector by Seamless Cloning and Optimization of Genetic Transformation System of Leaf Lettuce

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