基于CRISPR/Cas9加工番茄α-Man突变体的构建

doi:10.13560/j.cnki.biotech.bull.1985.2019-0134

摘要/Abstract

摘要：

为培育成熟果实软化程度低,货架期长的番茄植株,以加工番茄甘露糖苷酶基因（α-Man）为编辑对象,设计由番茄U6启动子驱动、长21 bp的guide RNA（gRNA）指导hCas9核酸酶,靶向编辑α-Man的第1个外显子。首先构建基于CRISPR/Cas9系统的植物表达载体,并通过农杆菌介导的遗传转化获得加工番茄转基因株系,然后取转基因番茄叶片基因组DNA,利用限制性内切酶法结合PCR扩增对α-Man编辑位点附近的DNA片段进行检测及测序分析。结果表明,14株转基因番茄植株有2株检测到突变现象。α-Man突变体TA克隆测序结果显示有2种编辑类型,一种表现为52 bp的缺失突变;另一种表现为单碱基突变。实现了对番茄α-Man的编辑。

关键词: 加工番茄, CRISPR/Cas9, α-Man, 果实成熟软化

Abstract:

In order to cultivate tomato variety with low-softening-degree and long-shelf-duration fruit,the first exon of the N-glycan processing enzyme gene（α-Man）was edited directly by the hCas9 nuclease combined with 21 bp guide RNA（gRNA）and while driven by tomato U6 promoter. Firstly,the plant expression vector based on CRISPR/Cas9 system was constructed,and the transgenic lines of tomato were obtained by Agrobacterium-mediated genetic transformation. Then the DNA fragment near α-Man’s editing site from the genomic DNA of transgenic tomato leaves was detected and sequenced by restriction endonuclease and PCR amplification. The results showed that 2 of 14 transgenic tomato strains were detected to have mutations. The sequencing results of the α-Man mutant TA clone suggested that there were 2 types of editing,one was a 52 bp deletion mutation and the other was a single base mutation. This indicates that the editing of tomato α-Man gene is achieved.

Key words: processed tomato, CRISPR/Cas9, α-Man, fruit ripening and softening

张圆圆, 邵冬南, 崔百明. 基于CRISPR/Cas9加工番茄α-Man突变体的构建[J]. 生物技术通报, 2019, 35(6): 9-16.

ZHANG Yuan-yuan, SHAO Dong-nan, CUI Bai-ming. Construction of Processing Tomato α-Man Mutant Based on CRISPR/Cas9[J]. Biotechnology Bulletin, 2019, 35(6): 9-16.

参考文献

[1] Consortium TTG.The tomato genome sequence provides insights into fleshy fruit evolution[J]. Nature, 2012, 485(7400):635.
[2] Cherian S, Figueroa C R, Nair H.‘Movers and shakers’ in the regulation of fruit ripening:a cross-dissection of climacteric versus non-climacteric fruit[J]. Journal of Experimental Botany, 2014, 65(17):4705-4722.
[3] Zhu M, Chen G, Zhou S, et al.A new tomato NAC(NAM/ATAF1/2/CUC2)transcription factor, SlNAC4, functions as a positive regul-ator of fruit ripening and carotenoid accumulation[J]. Plant & Cell Physiology, 2014, 55(1):119-135.
[4] Zhang LC, Zhu MK, Ren LJ, et al.The SlFSR gene controls fruit shelf-life in tomato[J]. Journal of Experimental Botany, 2018, 69(12):2897-2909.
[5] Roberts RM, Cetorelli JJ, Kirby EG, et al.Location of glycoproteins that contain glucosaminc in plant tissue[J]. Plant Physiology, 1972, 50(5):531-535.
[6] Handa AK, Singh NK, Biggs MS.Effect of tunicamycin on in vitro ripening of tomato pericarp tissue[J]. Physiologia Plantarum, 2010, 63(4):417-424.
[7] Priem B, Gitti R, Bush CA, et al.Structure of ten free N-glycans in ripening tomato fruit(arabinose is a constituent of a plant N-Glycan)[J]. Plant Physiology, 1993, 102(2):445-458.
[8] 罗川. 桃果实α-甘露糖苷酶基因的克隆及在软化过程中的表达分析[D]. 杨凌:西北农林科技大学, 2013.
[9] Irfan M, Ghosh S, Meli VS, et al.Fruit ripening regulation of α-mannosidase expression by the MADS box transcription factor RIPENING INHIBITOR and ethylene[J]. Front Plant Science, 2016, 7(26):10.
[10] Meli VS, Ghosh S, Prabha TN, et al.Enhancement of fruit shelf life by suppressing N-glycan processing enzymes[J]. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(6):2413-2418.
[11] Yang L, Huang W, Xiong F, et al.Silencing of SlPL, which encodes a pectate lyase in tomato, confers enhanced fruit firmness, prolonged shelf-life and reduced susceptibility to grey mould[J]. Plant Biotechnology Journal, 2017, 15(12):1544-1555.
[12] Han Y, Ban Q, Li H, et al.DkXTH8, A novel xyloglucan endotransglucosylase/hydrolase in persimmon, alters cell wall structure and promotes leaf senescence and fruit postharvest softening[J]. Scientific Reports, 2016, 6:39155.
[13] Uluisik S, Chapman NH, Smith R, et al.Genetic improvement of tomato by targeted control of fruit softening[J]. Nature Biotechnology, 2016, 34(9):950-952.
[14] 晁谨. 果实成熟和品质相关基因LeETR3/Lcyb共敲除载体构建及MDHAR的初步解析[D]. 合肥:合肥工业大学, 2017.
[15] Zhang F, Wen Y, Guo X.CRISPR/Cas9 for genome editing:progress, implications and challenges[J]. Human Molecular Genetics, 2014, 23(R1):R40.
[16] Chikako N, Narumi H, Sadao K, et al.Efficient genome editing in apple using a CRISPR/Cas9 system[J]. Sci Rep, 2016, 6:31481.
[17] Schaart JG, Wiel CCMVD, Lotz LAP, et al.Opportunities for products of new plant breeding techniques[J]. Trends in Plant Science, 2016, 21(5):438-449.
[18] Voytas DF, Gao C.Precision genome engineering and agriculture:Opportunities and regulatory challenges[J]. PLoS Biology, 2014, 12(6):e1001877.
[19] 王姗姗, 徐向军, 路浩, 等. α-甘露糖苷酶研究进展[J]. 动物医学进展, 2012, 33(1):92-97.
[20] Fu Y, Sander JD, Reyon D, et al.Improving CRISPR-Cas nuclease specificity using truncated guide RNAs[J]. Nature Biotechnology, 2014, 32:279-284.
[21] Wang W, Akhunova A, Chao S, et al.Optimizing multiplex CRISPR/Cas9-based genome editing for wheat[J]. Medical Mycology, 2018, 56(Suppl 2):S1-S159.
[22] Ito Y, Nishizawa-Yokoi A, Endo M, et al.CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening[J]. Biochemical & Biophysical Research Communications, 2015, 467(1):76-82.
[23] Shi J, Gao H, Wang H, et al.ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions[J]. Plant Biotechnology Journal, 2017, 15:207-216.
[24] Li Z, Liu Z, Xing A, et al.Cas9-guide RNA directed genome editing in soybean[J]. Plant Physiology, 2015, 169(2):960-970.
[25] 刘玟杉. 基于CRISPR/Cas9的拟南芥多基因编辑及其在基因功能研究中的应用[D]. 重庆:重庆大学, 2015.
[26] Xing HL, Dong L, Wang ZP, et al.A CRISPR/Cas9 toolkit for multiplex genome editing in plants[J]. BMC Plant Biology, 2014, 14(1):327.
[27] Li JF, Norville JE, Aach J, et al.Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9[J]. Nature Biotechnology, 2013, 31(8):688-691.
[28] 雷建峰, 伍娟, 陈晓俊, 等. 棉花花粉中高效转录U6启动子的克隆及功能分析[J]. 中国农业科学, 2015, 48(19):3794-3802.
[29] Chen X, Lu X, Shu N, et al.Targeted mutagenesis in cotton(Gossypium hirsutum L.)using the CRISPR/Cas9 system[J]. Scientific Reports, 2017, 7:44304.
[30] Sun X, Hu Z, Chen R, et al.Targeted mutagenesis in soybean using the CRISPR-Cas9 system[J]. Sci Rep, 2015, 5:10342.
[31] Cermak T, Curtin SJ, Gil-Humanes J, et al.A multipurpose toolkit to enable advanced genome engineering in plants[J]. The Plant Cell, 2017, 29(6):1196.