Construction of Expression Vector for Melon eIF4E Gene Knockout Using CRISPR-Cas9

doi:10.6048/j.issn.1001-4330.2018.05.005

Xinjiang Agricultural Sciences ›› 2018, Vol. 55 ›› Issue (5): 821-828.DOI: 10.6048/j.issn.1001-4330.2018.05.005

Previous Articles Next Articles

Construction of Expression Vector for Melon eIF4E Gene Knockout Using CRISPR-Cas9

YANG Jing¹, WANG Xu-hui^1,2, WANG Dong³, LI Guan¹

1.Bioengineering Research Center, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China;
2. Research Institutes of Bioenergy, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China;
3. Kashi University, Kashi Xinjiang 844006, China

Online:2018-05-20 Published:2018-07-25
Correspondence author: LI Guan(1949-), native place, Xinjiang, PhD, Doctoral supervisor, research field:Plant physiology,biochemistry and molecular biology. (E-mail) guanli@xju.Edu.cn
Supported by:
The National Natural Science Foundation of China"Gene digging and polymerization of powdery mildew resistance of melon"(31260258); the National Natural Science Foundation of China "Study on genetics and key regulatory factors of sugar accumulation in melon fruit"(31660297); Natural Science Foundation of Xinjiang Uygur Autonomous Region of China "Molecular selection of new germplasm for powdery mildew resistant and downy mildew of Hami melon"(2016D01C066)

CRISPR-Cas9技术敲除甜瓜eIF4E基因表达载体的构建

杨晶¹,王旭辉^1,2,王东³,李冠¹

1.新疆大学生物工程研究中心,乌鲁木齐 830046;
2.新疆农业科学院生物质能源研究所,乌鲁木齐 830091;
3.新疆喀什大学,新疆喀什 844006

通讯作者: 李冠(1949-),男,新疆人,教授,博士生导师,研究方向为植物生理生化与分子生物学,(E-mail)guanli@xju.Edu.cn
作者简介:杨晶(1992-),女,新疆人,硕士研究生,研究方向为生物化学与分子生物学,(E-mail)1605670957@qq.com
基金资助:
国家自然科学基金项目“甜瓜抗白粉病基因挖掘与聚合育种”(31260258);国家自然科学基金项目“甜瓜果实糖积累的遗传学与关键调控因子研究”(31660297);自治区自然科学基金项目“哈密瓜抗白粉病、霜霉病新种质的分子选育”(2016D01C066)

Abstract

Abstract: 【Objective】 The objective of this study is to construct gRNA expression vector of eIF4E gene in melon by using CRISPR-Cas9 plant gene editing system.【Method】 The gRNA primer was designed in the first exon region of eIF4E gene from "Queen" of melon in Xinjiang. The sgRNA cloning box was amplified by using the vector pP1C.4 as a template, the pP1C.4 vector was cut by EcoRI and XbaI enzymes, and the recombinant vector pP1C.4-eIF4E was constructed using DNA recombinase.【Result】 The results of colony PCR detection and sequencing showed that gRNA had been successfully linked into plant gene knockout vector pP1C.4.【Conclusion】 The plant gene knockout vector pP1C.4-eIF4E is constructed successfully and eIF4E can be directionally edited by pP1C.4-eIF4E, which is of great significance for further study on the function of eIF4E gene.

Key words: melon; CRISPR / Cas9; eIF4E; vector construction

摘要： 【目的】构建甜瓜eIF4E基因的CRISPR-Cas9植物基因编辑系统的gRNA 表达载体。为研究eIF4E基因功能奠定基础。【方法】以新疆主栽甜瓜皇后为材料,在eIF4E基因的第一个外显子区设计gRNA引物,以载体pP1C.4为模板,扩增获得sgRNA克隆框,使用EcoR I、Xba I双酶切pP1C.4载体,利用DNA重组酶构建重组载体pP1C.4-eIF4E。【结果】经菌落PCR检测和测序,gRNA 已经成功连接到植物基因敲除载体pP1C.4。【结论】构建了植物基因敲除载体pP1C.4-eIF4E,pP1C.4-eIF4E能对eIF4E基因进行定向编辑。

关键词: 甜瓜, CRISPR/Cas9, eIF4E , 载体构建

CLC Number:

S652
S503.52

YANG Jing, WANG Xu-hui, WANG Dong, LI Guan. Construction of Expression Vector for Melon eIF4E Gene Knockout Using CRISPR-Cas9[J]. Xinjiang Agricultural Sciences, 2018, 55(5): 821-828.

杨晶, 王旭辉, 王东, 李冠. CRISPR-Cas9技术敲除甜瓜eIF4E基因表达载体的构建[J]. 新疆农业科学, 2018, 55(5): 821-828.

References 21

[1]	王志丹. 中国甜瓜产业经济发展研究[D]. 北京:中国农业科学院博士论文,2014.WANG Zhi-dan. (2014). Research on Economic Development of Chinese Melon Industry[D]. PhD Dissertation. Chinese Academy of Agricultural Sciences, Beijing.
[2]	Ling, K. S., Levi, A., Adkins, S., Kousik, C. S., Miller, G., & Hassell, R., et al. (2013). Development and field evaluation of multiple virus-resistant bottle gourd ( lagenaria siceraria ). Plant Disease, 97(8): 1,057-1,062.
[3]	Rodríguez-Hernández, A. M., Gosalvez, B., Sempere, R. N., Burgos, L., And, M. A. A., & Truniger, V. (2012). Melon rna interference (rnai) lines silenced for cm-eif4e, show broad virus resistance. Molecular Plant Pathology, 13(7): 755-763.
[4]	Ruffel, S., Dussault, M. H., Palloix, A., Moury, B., Bendahmane, A., & Robaglia, C., et al. (2002). A natural recessive resistance gene against potato virus y in pepper corresponds to the eukaryotic initiation factor 4e (eif4e). Plant Journal, 32(6): 1,067-1,075.
[5]	Nicaise, V., German-Retana, S. R., Dubrana, M. P., & Mazier, M. (2003). The eukaryotic translation initiation factor 4e controls lettuce susceptibility to the potyvirus lettuce mosaic virus. Plant Physiology,132(3): 1,272-1,282.
[6]	Gao, Z., Johansen, E., Eyers, S., Thomas, C. L., Noel Ellis, T. H., & Maule, A. J. (2004). The potyvirus recessive resistance gene, sbm1, identifies a novel role for translation initiation factor eif4e in cell-to-cell trafficking. Plant Journal, 40(3): 376-385.
[7]	Ruffel, S., Gallois, J. L., Lesage, M. L., & Caranta, C. (2005). The recessive potyvirus resistance gene pot-1, is the tomato orthologue of the pepper pvr2-eif4e, gene. Molecular Genetics & Genomics, 274(4): 346-353.
[8]	Nieto, C., Morales, M., Orjeda, G., Clepet, C., Monfort, A., & Sturbois, B., et al. (2006). An eif4e allele confers resistance to an uncapped and non‐polyadenylated rna virus in melon. Plant Journal for Cell & Molecular Biology, 48(3): 452-462.
[9]	Duan, H., Richael, C., & Rommens, C. M. (2012). Overexpression of the wild potato eif4e-1 variant eva1 elicits potato virus y resistance in plants silenced for native eif4e-1. Transgenic Research, 21(5): 929-938.
[10]	廖鹏飞,聂旺,余雅心,等. ZFNs、TALENs 和 CRISPR-Cas 基因组靶向编辑技术及其在植物中的应用[J].基因组学与应用生物学,2016,35(2):442-451.LIAO Peng-fei,NIE Wang,YU Ya-xin,et al. (2016). ZFNs, TALENs and CRISPR-Cas Targeted Genome Editing Technologies and Their Applications in Plants [J]. Genomics and Applied Biology, 35(2):442-451. (in Chinese)
[11]	De, l. F. C., & Lu, T. K. (2017). Crispr-cas9 technology: applications in genome engineering, development of sequence-specific antimicrobials, and future prospects. Integrative Biology Quantitative Biosciences from Nano to Macro, 9(2): 109-122.
[12]	Gaj, T., Gersbach, C. A., & Iii, C. F. B. (2013). Zfn, talen and crispr/cas-based methods for genome engineering. Trends in Biotechnology, 31(7): 397-405.
[13]	Barrangou, R. (2014). Rna events. cas9 targeting and the crispr revolution. Science, 344(6185): 707-708.
[14]	景润春,卢洪.CRISPR/Cas9 基因组定向编辑技术的发展与在作物遗传育种中的应用[J].中国农业科学,2016,49(7):1 219-1 229.JING Run-chun, LU Hong. (2016). Development of CRISPR / Cas9 Genome Oriented Editing and Its Application in Crop Genetics and Breeding [J]. Scientia Agricultura Sinica, 49(7):1,219-1,229. (in Chinese)
[15]	Pyott, D. E., Sheehan, E., & Molnar, A. (2016). Engineering of crispr/cas9-mediated potyvirus resistance in transgene-free arabidopsis plants. Molecular Plant Pathology, 17(8): 1,276-1,288.
[16]	Chandrasekaran, J., Brumin, M., Wolf, D., Leibman, D., Klap, C., & Pearlsman, M., et al. (2016). Development of broad virus resistance in non-transgenic cucumber using crispr/cas9 technology. Molecular Plant Pathology, 17(7): 1,140-1,153.
[17]	Robaglia, C., & Caranta, C. (2006). Translation initiation factors: a weak link in plant rna virus infection. Trends in Plant Science, 11(1): 40-45.
[18]	Rodríguez-Hernández, A. M., Gosalvez, B., Sempere, R. N., Burgos, L., And, M. A. A., & Truniger, V. (2012). Melon rna interference (rnai) lines silenced for cm-eif4e, show broad virus resistance. Molecular Plant Pathology, 13(7): 755-763.
[19]	李君, 张毅, 陈坤玲, 等. CRISPR/Cas 系统: RNA 靶向的基因组定向编辑新技术[J]. 遗传, 2013, 35(11): 1 265-1 273.LI Juan, ZHANG Yi, CHEN Kun-ling, et al. (2013). CRISPR/Cas: a novel way of RNA-guided genome editing [J]. Yichuan (Hereditas) , 35(11): 1,265-1,273. (in Chinese)
[20]	Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., & Liang, Z., et al. (2013). Targeted genome modification of crop plants using a crispr-cas system. Nature Biotechnology, 31(8): 686-688.
[21]	Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J. D., & Kamoun, S. (2013). Targeted mutagenesis in the model plant nicotiana benthamiana using cas9 rna-guided endonuclease. Nature Biotechnology, 31(8): 691-693.

Construction of Expression Vector for Melon eIF4E Gene Knockout Using CRISPR-Cas9

CRISPR-Cas9技术敲除甜瓜eIF4E基因表达载体的构建

PDF

Knowledge

Abstract

Cite this article

share this article

References 21

Related Articles 15

Recommended Articles 0

Metrics

[1]	LI Junhua, MAO Jiancai, YANG Junyan, WANG Haojie. Analysis of the Current Situation of the Jiashi Melon Industry and discussion of Development Countermeasures [J]. Xinjiang Agricultural Sciences, 2024, 61(S1): 101-105.
[2]	ZHANG Hao, LIANG Qigan, ZHANG Xuejun, FU Xiaofa, CHEN Jihao, ZHOU Bo, HUANG Yuan. The resistance analysis of Cucumis metuliferus and its effect of grafting on melon quality [J]. Xinjiang Agricultural Sciences, 2024, 61(8): 1963-1968.
[3]	CHEN Jihao, ZHANG Hao, LIANG Qigan, FU Xiaofa, ZHANG Xuejun, MAO Jiancai. Effects of optimal fertilization and organic fertilizer application on the yield and quality of oriental melon [J]. Xinjiang Agricultural Sciences, 2024, 61(8): 1969-1975.
[4]	Kadierayi Maimaiti, ZHOU Tingting, HAN Sheng, Meilikehan Rexiti, Yushanjiang Maimaiti. Establishment of genetic transformation and regeneration systems for different melon varieties and rapid acquisition of gene edited plants [J]. Xinjiang Agricultural Sciences, 2024, 61(7): 1666-1672.
[5]	WU Fengyang, HUANG Wenqian, TIAN Xi, YANG Yulin. Research on recognition and localization of unordered stacked watermelons based on machine vision [J]. Xinjiang Agricultural Sciences, 2024, 61(7): 1805-1813.
[6]	YANG Junyan, YAN Miao, WU Haibo, YANG Wenli, WANG Haojie, MAO Jiancai, ZHAI Wenqiang, LI Junhua. The impact of high temperature on different thick -skinned melon varieties and comprehensive evaluation of its heat resistance [J]. Xinjiang Agricultural Sciences, 2024, 61(6): 1386-1396.
[7]	LIU Yang, ZHANG Zhengxiao, BAI Yujia, FENG Zuoshan. Effects of Alternaria alternata infection on active oxygen metabolism in different tissues of melon [J]. Xinjiang Agricultural Sciences, 2024, 61(6): 1397-1406.
[8]	MA Ling, SHEN Qi, KANG Qi, ZHANG Zhongxiang, JIA Hongtao, WANG Cheng. Change and association analysis of five heavy metals in melon plants at different growth stages [J]. Xinjiang Agricultural Sciences, 2024, 61(4): 892-899.
[9]	YANG Wenli, XU Lirong, LIU Bin, LING Yueming, LI Meihua, YANG Yong, FAN Rong, LI Yushun, ZHANG Yongbin, ZHANG Xuejun. Effects of salt stress on ion balance, membrane lipid peroxidation, and osmotic regulation substance accumulation in thin skin muskmelon ‘Huishu’ [J]. Xinjiang Agricultural Sciences, 2024, 61(4): 900-907.
[10]	LIANG Qigan, ZHANG Hao, HU Guozhi, CHEN Jihao, FENG Tongxin, CAO Qing, WANG Min, FU Xiaofa, YAN Miao, GAO Qiang, ZHANG Xuejun, ZHOU Bo, WANG Haojie. Growth,yield and quality of muskmelon in fertilization control facilities [J]. Xinjiang Agricultural Sciences, 2024, 61(3): 599-606.
[11]	ZHANG Wei, YANG Guohui, YU Hui. Effects of 2,4-epibrassinolide on growth and related genes expression of watermelon seedlings under drought Stress [J]. Xinjiang Agricultural Sciences, 2024, 61(3): 615-622.
[12]	LI Hui, BI Ying, WANG Xinyu, LEI Yaxin, ZHANG Qi, HUANG Shuai, Rezha kuwangdeke, WANG Jing. Effects of regulation of walnut green peel polyphenols on postharvest active oxygen metabolism and reduction of rotten in Hami melon [J]. Xinjiang Agricultural Sciences, 2024, 61(12): 2966-2975.
[13]	LI Chao, YANG Ying, ZHENG Heyun, YANG Jianli, CHEN Wei, YANG Mi, SUN Yuping. Genetic diversity and population structure analysis of melon germplasm resources in Xinjiang based on SSR fluorescence markers [J]. Xinjiang Agricultural Sciences, 2024, 61(11): 2614-2625.
[14]	SHEN Yue, LING Yueming, DUAN Xiaoyu, YANG Wenli, LI Meihua, WANG Yirou, WANG Huilin, ZHANG Xuejun. Development and verification of KASP marker for resistance to downy mildew in muskmelon [J]. Xinjiang Agricultural Sciences, 2024, 61(11): 2626-2634.
[15]	YAO Jun, QIN Yong, ZHENG Heyun, ZHANG Cuihuan, Zaituna Maimaiti, WANG Zhiwei, GEN Xinli. Development of composite plastic film of extract from Ficus carica leaves and the cling film application in keeping melon fresh [J]. Xinjiang Agricultural Sciences, 2024, 61(1): 118-126.