番茄不同截短U3启动子的克隆及功能分析
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摘要
从中蔬四号番茄品种中克隆能在番茄叶片中高效转录的SlU3启动子,为今后利用CRISPR/Cas9基因编辑技术进行分子育种奠定了基础。采用2轮PCR的方法,第1轮PCR从中蔬四号番茄品种中克隆了3种SlU3启动子,再采用Transfer PCR方法分别对3个启动子进行截短,共得到6个不同长度的启动子,并分别构建6个截短的SlU3启动子驱动GUS融合植物表达载体,利用农杆菌转化法分别转染番茄叶片。运用DNAMAN软件对已克隆的SlU3和拟南芥AtU3启动子序列具有转录功能的必要元件进行序列分析;经过2轮PCR,首先从中蔬四号番茄品种中克隆了3种SlU3-1P、SlU3-3P、SlU3-4P启动子,其长度分别是1 156,1 114,1 147 bp,再采用Transfer PCR方法分别对3个启动子进行截短,共得到6个不同长度的启动子,其长度依次是489,318,450,248,457,248 bp,并分别构建6个截短SlU3启动子驱动GUS融合植物表达载体,启动子序列比对分析发现,番茄U3启动子与拟南芥U3启动子一样,也含有比较保守的2个元件,USE和TATA框,2个元件之间的位置比较固定。利用农杆菌转化法分别转染番茄叶片,结果显示,成功侵染后的番茄叶片均被染成蓝色,表明已克隆的6种不同截短番茄SlU3启动子均具有转录活性。成功克隆了6种在番茄叶片中高效转录的SlU3启动子,为构建番茄CRISPR/Cas9基因编辑载体提供更多高效的内源启动子。
It is necessary for us to clone some endogenous promoters with high transcription activity in tomato leaves and construct CRISPR/Cas9 system,which could provide an effective technology for the research of molecular design breeding in tomato. Six different truncated tomato U3 promoters had cloned by two rounds of PCR amplification from tomato variety Zhongshu 4. Six GUS fusion expression vectors were constructed and transformed into tomato leaves by the agroinfiltration. After right sequencing of the transfer PCR products,the necessary element for transcriptional function of six kinds of SlU3 promoter was analyzed using DNAMAN software. The different truncated SlU3-1 P,SlU3-3 P and SlU3-4 P promoters from tomato variety Zhongshu 4 were performed to clone by two rounds of PCR amplification,altogether there were six different promoters. Lengths were 489,318,450,248,457,248 bp respectively. Six GUS fusion expression vectors were constructed and transformed into tomato leaves. The promoter sequence alignment analysis revealed that the tomato U3 promoter,like the Arabidopsis U3 promoter,also contained two more conserved elements,the USE and TATA boxes,and the positions between the two elements were relatively fixed. Transfected tomato leaves by agroinfiltration transformation method. The results showed that the six truncated U3 promoters could drive GUS expression in tomato leaves and tomato leaves could be stained blue and the shorter promoters also had stronger transcription activity as original promoters. Six different tomato U3 promoters had cloned with high-level transcription in tomato leaves,which could provide ideal endogenetic promoters for the construction of CRISPR/Cas9 system for tomato functional genomics research.
It is necessary for us to clone some endogenous promoters with high transcription activity in tomato leaves and construct CRISPR/Cas9 system,which could provide an effective technology for the research of molecular design breeding in tomato. Six different truncated tomato U3 promoters had cloned by two rounds of PCR amplification from tomato variety Zhongshu 4. Six GUS fusion expression vectors were constructed and transformed into tomato leaves by the agroinfiltration. After right sequencing of the transfer PCR products,the necessary element for transcriptional function of six kinds of SlU3 promoter was analyzed using DNAMAN software. The different truncated SlU3-1 P,SlU3-3 P and SlU3-4 P promoters from tomato variety Zhongshu 4 were performed to clone by two rounds of PCR amplification,altogether there were six different promoters. Lengths were 489,318,450,248,457,248 bp respectively. Six GUS fusion expression vectors were constructed and transformed into tomato leaves. The promoter sequence alignment analysis revealed that the tomato U3 promoter,like the Arabidopsis U3 promoter,also contained two more conserved elements,the USE and TATA boxes,and the positions between the two elements were relatively fixed. Transfected tomato leaves by agroinfiltration transformation method. The results showed that the six truncated U3 promoters could drive GUS expression in tomato leaves and tomato leaves could be stained blue and the shorter promoters also had stronger transcription activity as original promoters. Six different tomato U3 promoters had cloned with high-level transcription in tomato leaves,which could provide ideal endogenetic promoters for the construction of CRISPR/Cas9 system for tomato functional genomics research.
引文
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[21]雷建峰,徐新霞,代培红,李继洋,张巨松,刘晓东.不同截短U3启动子在棉花中的功能分析[J].棉花学报,2016,28(3):307-314. doi:10. 11963/issn.1002-7807. 201603015.Lei J F,Xu X X,Dai P H,Li J Y,Zhang J S,Liu XD. Functional analysis of different truncated U3 promot-ers in cotton[J]. Cotton Science,2016,28(3):307-314. doi:10. 11963/issn. 1002-7807. 201603015.
[2] Sun N,Zhao H M. Transcription activator-like effectornucleases(TALENs):A highly efficient and versatiletool for genome editing[J]. Biotechnology and Bioengi-neering,2013,110(7):1811-1821. doi:10. 1002/bit.24890.
[3] Chen K L,Gao C X. Targeted genome modification tech-nologies and their applications in crop improvements[J].Plant Cell Reports,2014,33(4,SI):575-583. doi:10.1007/s00299-013-1539-6.
[4] Feng Z,Zhang B,Ding W,Liu X,Yang D L,Wei P,Cao F,Zhu S,Zhang F,Mao Y,Zhu J K. Efficient ge-nome editing in plants using a CRISPR/Cas system[J].Cell Research,2013,23(10):1229-1232. doi:10.1038/cr. 2013. 114.
[5] Li J F,Norville J E,Aach J,Mccormack M,Zhang DD,Bush J,Church G M. Multiplex and homologous re-combination-mediated genome editing in Arabidopsis andNicotiana benthamiana using guide RNA and Cas9[J].Nature Biotechnology,2013,31(8):688-691. doi:10.1038/nbt. 2654.
[6] Janga M R,Campbell L M,Rathore K S. CRISPR/Cas9-mediated targeted mutagenesis in upland cotton(Gossypi-um hirsutum L.)[J]. Plant Molecular Biology,2017,94(4/5):349-360. doi:10. 1007/s11103-017-0599-3.
[7] Chen X G,Lu X K,Shu N,Wang S,Wang J J,WangD L,Guo L X,Ye W W. Targeted mutagenesis in cotton(Gossypium hirsutum L.)using the CRISPR/Cas9 system[J]. Scientific Reports,2017,7:44304. doi:10. 1038/srep44304.
[8] Li C,Unver T,Zhang B H. A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in Cotton(Gossypi-um hirsutum L.)[J]. Scientific Reports,2017,7:43902.doi:10. 1038/srep43902.
[9] Liang Z,Zhang K,Chen K L,Gao C X. Targeted muta-genesis in Zea mays using TALENs and the CRISPR/Cassystem[J]. Journal of Genetics and Genomics,2014,41(2):63-68. doi:10. 1016/j. jgg. 2013. 12. 001.
[10] Upadhyay S K,Kumar J,Alok A,Tuli R. RNA-Guidedgenome editing for target gene mutations in wheat[J].G3-Genes Genomes Genetics,2013,3(12):2233-2238. doi:10. 1534/g3. 113. 008847.
[11] Jacobs T B,Lafayette P R,Schmitz R J. Targeted ge-nome modifications in soybean with CRISPR/Cas9[J].BMC Biotechnology,2015,15(1):16. doi:10. 1186/s12896-015-0131-2.
[12] Shan Q,Wang Y,Li J,Gao C. Genome editing in riceand wheat using the CRISPR/Cas system[J]. NatureProtocols,2014,9(10):2395-2410. doi:10. 1038/nprot. 2014. 157.
[13] Cong L,Ran F A,Cox D,Lin S,Barretto R,HabibN,Hsu P D,Wu X,Jiang W,Marraffini L A,ZhangF. Multiplex genome engineering using CRISPR/Cassystems[J]. Science,2013,339(6121):819-823. doi:10. 1126/science. 1231143.
[14] Mali P,Yang L H,Esvelt K M,Aach J A,Dicarlo JE,Norville J E,Church G M. RNA-Guided human ge-nome engineering via Cas9[J]. Science,2013,339(6121):823-826. doi:10. 1126/science. 1232033.
[15] Li X,Jiang D H,Yong K L,Zhang D B. Varied tran-scriptional efficiencies of multiple Arabidopsis U6 smallnuclear RNA genes[J]. Journal of Integrative Plant Biol-ogy,2007,49(2):222-229.
[16] Zhang Z,Mao Y,Ha S,Liu W,Botella J R,Zhu J K.A multiplex CRISPR/Cas9 platform for fast and efficientediting of multiple genes in Arabidopsis[J]. Plant CellReports,2016,35(7):1519-1533. doi:10. 1007/s00299-015-1900-z.
[17] Erijman A,Shifman J M,Peleg Y. A single-tube assem-bly of DNA using the transfer-PCR(TPCR)platform[J]. DNA Cloning and Assembly Methods,2014,1116:89-101.
[18] Wiedenheft B,Sternberg S H,Doudna J A. RNA-guidedgenetic silencing systems in bacteria and archaea[J].Nature,2012,482(7385):331-338. doi:10. 1038/na-ture10886.
[19] Sugano S S,Shirakawa M,Takagi J,Matsuda Y,Shi-mada T,Hara-Nishimura I,Kohchi T. CRISPR/Cas9-Mediated targeted mutagenesis in the liverwort Marchan-tia polymorpha L.[J]. Plant and Cell Physiology,2014,55(3):475-481. doi:10. 1093/pcp/pcu014.
[20] Marshallsay C,Kiss T,Filipowicz W. Amplification ofplant u3 and u6 snrna gene-sequences using primersspecific for an upstream promoter element and conservedintragenic regions[J]. Nucleic Acids Research,1990,18(12):3459-3466. doi:10. 1093/nar/18. 12. 3459.
[21]雷建峰,徐新霞,代培红,李继洋,张巨松,刘晓东.不同截短U3启动子在棉花中的功能分析[J].棉花学报,2016,28(3):307-314. doi:10. 11963/issn.1002-7807. 201603015.Lei J F,Xu X X,Dai P H,Li J Y,Zhang J S,Liu XD. Functional analysis of different truncated U3 promot-ers in cotton[J]. Cotton Science,2016,28(3):307-314. doi:10. 11963/issn. 1002-7807. 201603015.