CRISPR/Cas9 Technology for RcPDS1 Gene Editing in Rehmannia chingii

doi:10.16420/j.issn.0513-353x.2021-0475

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (7): 1532-1544.doi: 10.16420/j.issn.0513-353x.2021-0475

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CRISPR/Cas9 Technology for RcPDS1 Gene Editing in Rehmannia chingii

ZUO Xin¹, LI Mingming¹, LI Xinrong¹, MIAO Chunyan¹, LI Yanfang¹, YANG Xu¹, ZHANG Zhongyi², WANG Fengqing¹^,^*()

¹College of Agronomy,Henan Agricultural University,Zhengzhou 450046,China
²College of Agriculture,Fujian Agriculture and Forestry University,Fuzhou 350002,China

Received:2021-11-15 Revised:2022-03-15 Online:2022-07-25 Published:2022-07-29
Contact: WANG Fengqing E-mail:heauzycxw@126.com

Abstract

Abstract:

To study the application of CRISPR/Cas9 technology in Rehmannia chingii,the phytoene desaturase gene（RcPDS1）of R. chingii was cloned,and its cDNA sequence and genomic DNA sequence were amplified by polymerase chain reaction（PCR）. The constructed single-target CRISPR/Cas9 plasmid was transformed into R. chingii genome using Agrobacterium-mediated genetic transformation method,and the types of gene editing were analyzed by TA clone sequencing. The results showed that the full-length cDNA sequence of RcPDS1 was obtained. The cDNA sequence of RcPDS1 with a length of 1 743 bp contains a single open reading frame,which encoding 580 amino acid residues. The length of genomic DNA sequence of RcPDS1 gene is 8 041 bp,which contains 14 introns and 15 exons. A total of 57 transgenic lines were obtained through A. tumefaciens-mediated genetic transformation,of which 20 lines（35.09%）had the obvious albino phenotype. The results of sequence analysis showed that the target sites mutation modes of RcPDS1 were mainly deletions,substitutions and insertions. It showed that the CRISPR/Cas9 system is a powerful tool to achieve targeted knockout of target genes in R. chingii genome.

Key words: Rehmannia chingii, CRISPR/Cas9, RcPDS1, gene editing

CLC Number:

Q78
R 282

ZUO Xin, LI Mingming, LI Xinrong, MIAO Chunyan, LI Yanfang, YANG Xu, ZHANG Zhongyi, WANG Fengqing. CRISPR/Cas9 Technology for RcPDS1 Gene Editing in Rehmannia chingii[J]. Acta Horticulturae Sinica, 2022, 49(7): 1532-1544.

Figures/Tables 10

Table 1 Primer names and sequences

用途 Aim	产物长度/bp Product size	引物名称 Primer name	引物序列（5′-3′） Primer sequence
RcPDS1扩增 Amplification of RcPDS1	6 781（DNA） 1 882（cDNA）	RcPDS1_F	CAGTTGCCTGAGCTGTTGAA
RcPDS1扩增 Amplification of RcPDS1	6 781（DNA） 1 882（cDNA）	RcPDS1_R	GCTTCCCTATCTTCTGTCTTCC
RcPDS1 qRT-PCR	103	RcPDS1_qF	TATCATTTGCAGTTAGTG
RcPDS1 qRT-PCR	103	RcPDS1_qR	ACAACTTTCAAAGGGATTGC
靶序列的合成 Construction of target site	19	sgRcPDS1_F	AAGCAAGGGATGTGCTGGG
靶序列的合成 Construction of target site	19	sgRcPDS1_R	CCCAGCACATCCCTTGCTT
转基因鉴定 Identification of the transformation	474	Cas9_F	TCAACGGCATTCGGGACAAG
	474	Cas9_R	CCACATACATATCGCGGCCA
	281	pKSE401_F	TGTCCCAGGATTAGAATGATTAGGC
	281	sgRcPDS1_R	CCCAGCACATCCCTTGCTT
靶位点序列扩增 Amplification of target region	1 387	RcPDS1_TF	CTTCTCCTCGTCCAAACAAG
靶位点序列扩增 Amplification of target region	1 387	RcPDS1_TR	AGCTCTCCAAACAGGTTCTG
Gap序列扩增 Amplification of the gap sequence	828	PDSgap1_F	CAGCGAAAACAAGCTGAATCTG
	828	PDSgap1_R	GATTTCCTCCAAACTAACCGTG
		PDSgap2_F	TCGGACATGTTTCTGCTATCAA
	2 091	PDSgap2_R	TCCTTCCATTGCAACCGATC
	1 012	PDSgap3_F	ACAAACCAGGAGAGTTCAGC
	1 012	PDSgap3_R	GCTTCAACATAAGACTGACCG
	278	PDSgap4_F	CCTGGTACCGAACCTTGTC
	278	RcPDS1_R	GCTTCCCTATCTTCTGTCTTCC

Fig. 1 Gel electrophoresis diagram of PCR amplified products of cDNA（A）and DNA（B）of RcPDS1 gene

Fig. 2 Alignment of RcPDS1 and AtPDS3

Fig. 3 Phylogenetic tree of RcPDS1

Fig. 4 The expression level of RcPDS1 gene in different tissues of Rehmannia chingii

Fig. 5 CRISPR/Cas9 target site selection and recombinant plasmid constructionA:Schematic diagram showing RcPDS1 gene structure;B:Result of transformation detection by PCR;C:Schematic of the CRISPR/Cas9 binary vector pKSE401.

Fig. 6 Genetic transformation of Rehmannia chingii and albino phenotype mutantsA:Embryogenic calluses were cultured on the medium with kanamycine;B:Positive albino callus;C:Positive albino shoot;D:Positive albino shoot and green shoot regenerated from the same callus;E:Positive albino shoot cultured under light;F:Browning albino shoots;G:Fully albino shoot;H:Chimeric albino shoot.

Fig. 7 Positive identification of transgenic plants

Fig. 8 The phenotype of wild type and RcPDS1 knockout mutants plants of Rehmannia chingii

Fig. 9 RcPDS1 target mutations in transgenic plants of Rehmannia chingiiGreen letters are insertions,pink letters are substitutions,dashes are deletions,and the number at the right-hand side represent types of base mutation.

References 41

[1]	Alagoz Y, Gurkok T, Zhang B, Unver T. 2016. Manipulating the biosynthesis of bioactive compound alkaloids for next-generation metabolic engineering in opium poppy using CRISPR-Cas 9 genome editing technology. Scientific Reports, 6 (1):30910. doi: 10.1038/srep30910 URL
[2]	Bai C, Capell T, Berman J, Medina V, Sandmann G, Christou P, Zhu C F. 2016. Bottlenecks in carotenoid biosynthesis and accumulation in rice endosperm are influenced by the precursor-product balance. Plant Biotechnology Journal, 14 (1):195-205. doi: 10.1111/pbi.12373 URL
[3]	Beying N, Schmidt C, Pacher M, 2020.CRISPR-Cas9-mediated induction of heritable chromosomal translocations in Arabidopsis. Nature Plants, 6 (6):638-645. doi: 10.1038/s41477-020-0663-x URL
[4]	Cai Y P, Wang L W, Chen L, Wu T T, Liu L P, Sun S, Wu C X, Yao W W, Jiang B J, Yuan S, Han T F, Hou W S. 2020. Mutagenesis of GmFT2a and GmFT5a mediated by CRISPR/Cas 9 contributes for expanding the regional adaptability of soybean. Plant Biotechnology Journal, 18 (1):298-309. doi: 10.1111/pbi.13199 URL
[5]	Cazzonelli C I, Pogson B J. 2010. Source to sink:regulation of carotenoid biosynthesis in plants. Trends in Plant Science, 15 (5):266-274. doi: 10.1016/j.tplants.2010.02.003 URL
[6]	Chen Duanfen, Peng Zhenhua, Gao Zhimin. 2008. Cloning and expression analysis of PDS gene in Narcissus tazetta var.chinensis. Molecular Plant Breeding, 6 (3):574-578. (in Chinese)
	陈段芬, 彭镇华, 高志民. 2008. 中国水仙八氢番茄红素脱氢酶基因(PDS)的克隆及表达分析. 分子植物育种, 6 (3):574-578.
[7]	Chen K L, Wang Y P, Zhang R, Zhang H W, Gao C X. 2019. CRISPR/Cas genome editing and precision plant breeding in agriculture. Annual Review of Plant Biology, 70 (1):667-697. doi: 10.1146/annurev-arplant-050718-100049 URL
[8]	Chen Y T, Mao W W, Liu T, Feng Q Q, Li L, Li B B. 2020. Genome editing as a versatile tool to improve horticultural crop qualities. Horticultural Plant Journal, 6 (6):372-384. doi: 10.1016/j.hpj.2020.11.004 URL
[9]	Deng C P, Shi M, Fu R, Zhang Y, Wang Q, Zhou Y, Wang Y, Ma X Y, Kai G Y. 2020. An ABA-responsive SmbZIP 1 is involved in modulating biosynthesis of phenolic acids and tanshinones in Salvia miltiorrhiza. Journal of Experimental Botany, 71 (19):5948-5962. doi: 10.1093/jxb/eraa295 URL
[10]	Deng C Y, Zhang F, Wang J Y, Li Y F, Huang H, Dai S L. 2021. Tobacco rattle virus-induced phytoene desaturase(PDS)silencing in Centaurea cyanus. Horticultural Plant Journal, 7 (2):159-166. doi: 10.1016/j.hpj.2020.08.002 URL
[11]	Feng Z Y, Zhang B T, Ding W N, Liu X D, Yang D L, Wei P L, Gao F Q, Zhu S H, Zhang F, Mao Y F, Zhu J K. 2013. Efficient genome editing in plants using a CRISPR/Cas system. Cell Research, 23 (10):1229-1232. doi: 10.1038/cr.2013.114 URL
[12]	Kui L, Chen H T, Zhang W X, He S M, Xiong Z J, Zhang Y S, Yan L, Zhong C F, He F M, Chen J W, Zeng P, Zhang G H, Yang S C, Dong Y, Wang W, Cai J. 2017. Building a genetic manipulation tool box for orchid biology:identification of constitutive promoters and application of CRISPR/Cas9 in the orchid,Dendrobium officinale. Frontiers in Plant Science, 7 (30):2036.
[13]	Kumagai M H, Donson J, Della C G, Harvey D, Hanley K, Grill L K. 1995. Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proceedings of the National Academy of Sciences of the United States of America, 92:1679-1683.
[14]	Li J F, Norville J E, Aach J, McCormack M, Zhang D D, Bush J, Church G M, Sheen J. 2013. Multiplex and homologous recombination mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature Biotechnology, 31 (8):688-691. doi: 10.1038/nbt.2654 URL
[15]	Li T D, Yang X P, Yu Y, Si X M, Zhai X W, Zhang H W, Dong W X, Gao C X, Xu C. 2018. Domestication of wild tomato is accelerated by genome editing. Nature Biotechnology, 36:1160-1163. doi: 10.1038/nbt.4273 URL
[16]	Liu Yanfei, Shi Guoru, Wang Xin, Zhang Chunlei, Wang Yan, Chen Ruoyun, Yu Dequan. 2016. Chemical constituents from whole plants of Rehmannia chingii. Chinese Traditional and Herbal Drugs, 47 (11):1830-1833. (in Chinese)
	刘彦飞, 史国茹, 王欣, 张春磊, 王艳, 陈若芸, 于德泉. 2016. 天目地黄化学成分研究. 中草药, 47 (11):1830-1833.
[17]	Ma C F, Liu M C, Li Q F, Si J, Ren X S, Song H Y. 2019. Efficient BoPDS gene editing in cabbage by the CRISPR/Cas9 system. Horticultural Plant Journal, 5 (4):164-169. doi: 10.1016/j.hpj.2019.04.001 URL
[18]	Ma Dandan, Xia Guohua, Jin Feifei, Li Genyou. 2010. Study on regeneration system from callus of Rehmannia chingii f. albiflora. Guihaia, 30 (5):686-690. (in Chinese)
	马丹丹, 夏国华, 金翡翡, 李根有. 2010. 白花天目地黄的愈伤组织再生体系研究. 广西植物, 30 (5):686-690.
[19]	Matsumura H, Shiomi K, Yamamoto A, Taketani Y, Fukayama H. 2020. Hybrid rubisco with complete replacement of rice rubisco small subunits by sorghum counterparts confers C₄ plant-like high catalytic activity. Molecular Plant, 13 (11):1570-1581. doi: 10.1016/j.molp.2020.08.012 URL
[20]	McQuinn R P, Wong B, Giovannoni J J. 2018. AtPDS overexpression in tomato: exposing unique patterns of carotenoid self-regulation and an alternative strategy for the enhancement of fruit carotenoid content. Plant Biotechnology Journal, 16 (2):482-494. doi: 10.1111/pbi.12789 pmid: 28703352
[21]	Meng Xiongyu. 2015. Studies on chemistry composition、standard of quality and anti-senescence activites of Rehmannia chingii Li.[M. D. Dissertation]. Yinchuan: Ningxia Medical University. (in Chinese)
	蒙雄裕. 2015. 天目地黄化学成分、质量标准及其抗衰老活性研究[硕士论文]. 银川: 宁夏医科大学.
[22]	Mussolino C, Cathomen T. 2013. RNA guides genome engineering. Nature Biotechnology, 31 (3):208-209. doi: 10.1038/nbt.2527 pmid: 23471067
[23]	Naim F, Dugdale B, Kleidon J, Brinin A, Shand K, Water-house P, Dale J. 2018. Gene editing the phytoene de-saturase alleles of Cavendish banana using CRISPR/Cas9. Transgenic Research, 27 (5):451-460. doi: 10.1007/s11248-018-0083-0 URL
[24]	Nishitani C, Hirai N, Komori S, Wada M, Okada K, Osakabe K, Yamamoto T, Osakabe Y. 2016. Efficient genome editing in apple using a CRISPR/Cas9 system. Scientific Reports, 6 (1):31481. doi: 10.1038/srep31481 URL
[25]	Osakabe Y, Liang Z C, Ren C, Nishitani C, Osakabe K, Wada Masato, Komori S, Malnoy M, Velasco R, Poli M, Jung M, Koo O, Viiola R, Kanchiswamy C N. 2018. CRISPR-Cas9-mediated genome editing in apple and grapevine. Nature Protocols, 13 (12):2844-2863. doi: 10.1038/s41596-018-0067-9 pmid: 30390050
[26]	Schachtsiek J, Stehle F. 2019. Dataset on nicotine-free,nontransgenic tobacco(Nicotiana tabacum L)edited by CRISPR-Cas9. Data in Brief, 26:104395. doi: 10.1016/j.dib.2019.104395 URL
[27]	Shen J S, Si W J, Wu Y T, Xu Y, Wang J, Cheng T R, Zhang Q X, Pan H T. 2021. Establishment and verification of an efficient virus-induced gene silencing system in Forsythia. Horticultural Plant Journal, 7 (1):81-88. doi: 10.1016/j.hpj.2020.09.001 URL
[28]	Tian L. 2015. Recent advances in understanding carotenoid-derived signaling molecules in regulating plant growth and development. Frontiers in Plant Science, 6:790. doi: 10.3389/fpls.2015.00790 pmid: 26442092
[29]	Wang Dan, Wang Mi, Liu Jun, Zhou Xiaohui, Liu Songyu, Yang Yan, Zhuang Yong. 2022. Cloning of U 6 promoters and establishment of CRISPR/Cas9 mediated gene editing system in eggplant. Acta Horticulturae Sinica, 49 (4):791-800. (in Chinese)
	王丹, 王谧, 刘军, 周晓慧, 刘松瑜, 杨艳, 庄勇. 2022. 茄子U6启动子克隆及CRISPR/Cas9介导的基因编辑体系建立. 园艺学报, 49 (4):791-800.
[30]	Wang H, Wu Y, Zhang Y, Yang J, Fan W, Zhang H, Zhao S, Yuan L, Zhang P. 2019. CRISPR/Cas9-Based Mutagenesis of starch biosynthetic genes in sweet potato(Ipomoea batatas)for the improvement of starch quality. International Journal of Molecular Sciences, 20 (19):4702. doi: 10.3390/ijms20194702 URL
[31]	Wu Wen, Zhao Nan, Li Hongqing, Qu Weijing. 2006. Distribution and accumulation trends of catalpol in resource species of Rehmannia. Journal of East China Normal University(Natural Science),(4):91-96. (in Chinese)
	武雯, 赵楠, 李宏庆, 瞿伟菁. 2006. 地黄属资源植物中梓醇的分布及积累动态. 华东师范大学学报(自然科学版),(4):91-96.
[32]	Xing H L, Dong L, Wang Z P, Han C Y, Liu B, Wang X C, Chen Q J. 2014. A CRISPR/Cas 9 toolkit for multiplex genome editing in plants. BMC Plant Biology, 14 (1):327. doi: 10.1186/s12870-014-0327-y URL
[33]	Yang Feng, Yang Qinsong, Gao Yuhao, Ma Yunjing, Xu ying, Teng Yuanwen, Bai songling. 2021. Establishment of dual-cut CRISPR/Cas 9 gene editing system in pear calli. Acta Horticulturae Sinica, 48 (5):873-882. (in Chinese)
	杨锋, 杨钦淞, 高雨豪, 马云晶, 许英, 滕元文, 白松龄. 2021. 梨愈伤组织双靶点CRISPR/Cas9基因编辑系统的建立. 园艺学报, 48 (5):873-882.
[34]	Yang Lushan, Guo Ye, Hu Yang, Wen Yingqiang. 2020. CRISPR/Cas9-mediated mutagenesis of VviEDR2 results in enhanced resistance to powdery mildew in Grapevine(Vitis vinifera). Acta Horticulturae Sinica, 47 (4):623-634. (in Chinese)
	杨禄山, 郭晔, 胡洋, 文颖强. 2020. 利用CRISPR/Cas9系统敲除葡萄中VviEDR2提高对白粉病的抗性. 园艺学报, 47 (4):623-634.
[35]	Zhang R X, Li M X, Jia Z P. 2008. Rehmannia glutinosa:review of botany,chemistry and pharmacology. Journal of Ethnopharmacology, 117 (2):199-214. doi: 10.1016/j.jep.2008.02.018 URL
[36]	Zhao Le, Zhu Yunhao, Wang Min, Han Yongguang, Ma Lili, Feng Weisheng, Zheng Xiaoke. 2021. Estimation of Rehmannia glutinosa genome size based on flow cytometry and genome survey analysis. Chinese Traditional and Herbal Drugs, 52 (3):821-826. (in Chinese)
	赵乐, 朱畇昊, 王敏, 韩永光, 马利利, 冯卫生, 郑晓珂. 2021. 基于流式细胞术和基因组survey分析的地黄基因组研究. 中草药, 52 (3):821-826.
[37]	Zheng C J, Wu Y, Zhu J Y, Qin L P. 2014. Chemical constituents of the aerial parts of Rehmannia chingii. Chemistry of Natural Compounds, 50 (3):560-561. doi: 10.1007/s10600-014-1017-6 URL
[38]	Zhou P, Peng J Y, Zeng M J, Wu L X, Fan Y X, Zeng L H. 2021. Virus-induced gene silencing(VIGS)in Chinese narcissus and its use in functional analysis of NtMYB3. Horticultural Plant Journal, 7 (6):565-572. doi: 10.1016/j.hpj.2021.04.009 URL
[39]	Zhou Z, Tan H, Li Q, Chen J, Gao S, Wang Y, Chen W, Zhang L. 2018. CRISPR/Cas9-mediated efficient targeted mutagenesis of RAS in Salvia miltiorrhiza. Phytochemistry, 148:63-70. doi: 10.1016/j.phytochem.2018.01.015 URL
[40]	Zhu H C, Li C, Gao C. 2020. Applications of CRISPR-Cas in agriculture and plant biotechnology. Nature Reviews Molecular Cell Biology, 21 (12):661-677. doi: 10.1038/s41580-020-00288-9 URL
[41]	Zuo X, Wang F Q, Li X R, Li M M. 2020. Transcriptome-based screening and the optimal reference genes for real-time quantitative PCR in Rehmannia chingii and R. henryi. Biologia Plantarum, 64:798-806. doi: 10.32615/bp.2020.154 URL

CRISPR/Cas9 Technology for RcPDS1 Gene Editing in Rehmannia chingii

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