Cloning of U6 Promoters and Establishment of CRISPR/Cas9 Mediated Gene Editing System in Eggplant

doi:10.16420/j.issn.0513-353x.2020-0265

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (4): 791-800.doi: 10.16420/j.issn.0513-353x.2020-0265

• Research Papers • Previous Articles Next Articles

Cloning of U6 Promoters and Establishment of CRISPR/Cas9 Mediated Gene Editing System in Eggplant

WANG Dan¹^,², WANG Mi¹^,², LIU Jun², ZHOU Xiaohui², LIU Songyu², YANG Yan¹^,²^,^*(), ZHUANG Yong¹^,²^,^*()

1. College of Horticulture,Nanjing Agricultural University,Nanjing 210095,China
2. Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement,Institute of Vegetable Crops,Jiangsu Academy of Agricultural Sciences, Nanjing 210014,China

Received:2021-10-20 Revised:2022-01-29 Online:2022-04-25 Published:2022-04-24
Contact: YANG Yan,ZHUANG Yong E-mail:yzyangyan890618@163.com;jaaszy@163.com

Abstract

Abstract:

Seven U6 RNAs were identified from the eggplant（Solanum melongena L.）genome based on the conserved U6 RNA sequence in Arabidopsis and the responding promoters were cloned by PCR amplification. GUS staining results showed that four SmU6 promoters（SmU6-1P,SmU6-2P,SmU6-4P,SmU6-7P）were capable of transcriptional activity. The CRISPR/Cas9 vectors with AtU6-P/SmU6-1P driving SmWRKY4 sgRNA were constructed. T₀ plants were obtained by Agrobacterium-mediated transformation of eggplant cotyledons. The DNA of T₀ plants were extracted to amplify the target fragments,which were subsequently sequenced to analyze whether SmWRKY4 gene were edited or not. As a result,the CRISPR/Cas9 vector containing SmU6-1P could edit SmWRKY4 with an editing rate of 27.0%. However,no mutation was detected in T₀ plants based on AtU6-P. These results indicated that the editing efficiency of eggplant U6 promoter was better than that of Arabidopsis U6 promoter in eggplant genetic transformation.

Key words: eggplant, U6 promoter, CRISPR/Cas9, gene editing

CLC Number:

S641.1

WANG Dan, WANG Mi, LIU Jun, ZHOU Xiaohui, LIU Songyu, YANG Yan, ZHUANG Yong. Cloning of U6 Promoters and Establishment of CRISPR/Cas9 Mediated Gene Editing System in Eggplant[J]. Acta Horticulturae Sinica, 2022, 49(4): 791-800.

Figures/Tables 7

References 30

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引物名称 Primer name	上游引物序列（5′-3′） Forward sequence	下游引物序列（5′-3′） Reverse sequence
SmU6-1P	CCCAAGCTTGACAACATCTGCCATTGGA	CGGGATCCGAACTCATTACTTCGCTAGG
SmU6-2P	CCCAAGCTTTCTCATTGTATTGCGTCGAA	CGGGATCCGAACTCCTTTACTTCGCTAA
SmU6-3P	CCCAAGCTTACGAACCAATTGATGCACAC	CGGGATCCGAACTCATTACTTCGCTAGG
SmU6-4P	CCCAAGCTTGTACTAGATCAGCGAGATC	CGGGATCCGAACTCCAATACTTCGCTAG
SmU6-5P	CCCAAGCTTCACGATGCCCTAT	CGGGATCCCGATACGTAACGCCTGGC
SmU6-6P	CCCAAGCTTATCCTATCAATGAAAGCTCC	CGGGATCCCAATGGATTAATTACTCACTGGTGA
SmU6-7P	CCCAAGCTTCTAGGCTAAAGTAGGGCAA	CGGGATCCAAGCGCTAAAGTAAGGTTCGGCC
AtU6-P	CCCAAGCTTCATTCGGAGTTTTTGTATCTTG	CGGGATCCCAATCACTACTTCGACTCTAGCT
SmU6-1P-a	CGGAATTCGACAACATCTGCCATTGGA	GCTCTAGAGAACTCATTACTTCGCTAGG
SmU6-1P-b	CAGGAAACAGCTATGACCATATTCGACAACATCTGCCATTGG	GCTATTTCTAGCTCTAAAACGTACGGCTGATTTGGAGCTGCGAACTCATTACTTCGCTA
AtU6-P-b	CAGGAAACAGCTATGACCATATTCATTCGGAGTTTTTGTATC	GCTATTTCTAGCTCTAAAACGTACGGCTGATTTGGAGCTGCAATCACTACTTCGACTCT
RTCas9	AAGCCCATCAGAGAGCAGG	TGTCGCCTCCCAGCTGAG
WRKY4test	CGGCATTGAACAGTACCAGA	CCAGATGTCAGCCTCCATTT

引物名称 Primer name	上游引物序列（5′-3′） Forward sequence	下游引物序列（5′-3′） Reverse sequence
SmU6-1P	CCCAAGCTTGACAACATCTGCCATTGGA	CGGGATCCGAACTCATTACTTCGCTAGG
SmU6-2P	CCCAAGCTTTCTCATTGTATTGCGTCGAA	CGGGATCCGAACTCCTTTACTTCGCTAA
SmU6-3P	CCCAAGCTTACGAACCAATTGATGCACAC	CGGGATCCGAACTCATTACTTCGCTAGG
SmU6-4P	CCCAAGCTTGTACTAGATCAGCGAGATC	CGGGATCCGAACTCCAATACTTCGCTAG
SmU6-5P	CCCAAGCTTCACGATGCCCTAT	CGGGATCCCGATACGTAACGCCTGGC
SmU6-6P	CCCAAGCTTATCCTATCAATGAAAGCTCC	CGGGATCCCAATGGATTAATTACTCACTGGTGA
SmU6-7P	CCCAAGCTTCTAGGCTAAAGTAGGGCAA	CGGGATCCAAGCGCTAAAGTAAGGTTCGGCC
AtU6-P	CCCAAGCTTCATTCGGAGTTTTTGTATCTTG	CGGGATCCCAATCACTACTTCGACTCTAGCT
SmU6-1P-a	CGGAATTCGACAACATCTGCCATTGGA	GCTCTAGAGAACTCATTACTTCGCTAGG
SmU6-1P-b	CAGGAAACAGCTATGACCATATTCGACAACATCTGCCATTGG	GCTATTTCTAGCTCTAAAACGTACGGCTGATTTGGAGCTGCGAACTCATTACTTCGCTA
AtU6-P-b	CAGGAAACAGCTATGACCATATTCATTCGGAGTTTTTGTATC	GCTATTTCTAGCTCTAAAACGTACGGCTGATTTGGAGCTGCAATCACTACTTCGACTCT
RTCas9	AAGCCCATCAGAGAGCAGG	TGTCGCCTCCCAGCTGAG
WRKY4test	CGGCATTGAACAGTACCAGA	CCAGATGTCAGCCTCCATTT

Cloning of U6 Promoters and Establishment of CRISPR/Cas9 Mediated Gene Editing System in Eggplant

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