茄子U6启动子克隆及CRISPR/Cas9介导的基因编辑体系建立

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

摘要/Abstract

摘要：

利用拟南芥（Arabidopsis thaliana）U6 RNA保守序列从茄子（Solanum melongena L.）基因组中鉴定到7个U6 RNA,并采用PCR技术成功克隆其启动子序列。GUS染色结果表明4个茄子U6启动子（SmU6-1P、SmU6-2P、SmU6-4P和SmU6-7P）具有转录活性。构建以SmU6-1P/AtU6-P驱动SmWRKY4 sgRNA的CRISPR/Cas9基因编辑载体,通过农杆菌介导侵染茄子子叶外植体得到T₀代植株。以T₀代植株的DNA为模板扩增WRKY4片段并对产物测序,测序结果显示以SmU6-1P构建的CRISPR/Cas9载体能靶向编辑SmWRKY4,编辑率为27.0%。在基于AtU6-P的T₀代植株中未检测到碱基突变,表明在茄子遗传转化中,茄子SmU6启动子编辑效率高于拟南芥AtU6启动子。

关键词: 茄子, U6启动子, CRISPR/Cas9, 基因编辑

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

中图分类号:

S641.1

王丹, 王谧, 刘军, 周晓慧, 刘松瑜, 杨艳, 庄勇. 茄子U6启动子克隆及CRISPR/Cas9介导的基因编辑体系建立[J]. 园艺学报, 2022, 49(4): 791-800.

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.

图/表 7

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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