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

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

Abstract

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

CLC Number:

S636.2

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.

Figures/Tables 10

References 50

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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	-