植物学报 ›› 2023, Vol. 58 ›› Issue (6): 905-916.DOI: 10.11983/CBB22250
苗春妍1,†, 李铭铭1,†, 左鑫1, 丁宁1, 杜家方1, 李娟1, 张重义2, 王丰青1,*()
收稿日期:
2022-10-21
接受日期:
2023-03-17
出版日期:
2023-11-01
发布日期:
2023-11-27
通讯作者:
* E-mail: fqwang@henau.edu.cn
作者简介:
† 共同第一作者
基金资助:
Chunyan Miao1,†, Mingming Li1,†, Xin Zuo1, Ning Ding1, Jiafang Du1, Juan Li1, Zhongyi Zhang2, Fengqing Wang1,*()
Received:
2022-10-21
Accepted:
2023-03-17
Online:
2023-11-01
Published:
2023-11-27
Contact:
* E-mail: fqwang@henau.edu.cn
About author:
† These authors contributed equally to this paper
摘要: 湖北地黄(Rehmannia henryi)是一种具有重要价值的药用植物, 其基因编辑研究还未见报道。为建立湖北地黄基因编辑体系, 克隆了类胡萝卜素合成途径中八氢番茄红素脱氢酶(PDS)的编码基因, 并构建RhPDS1基因的CRISPR/Cas9载体, 以根癌农杆菌介导法转化湖北地黄基因组。结果表明, RhPDS1转录本含1 764 bp的开放阅读框(ORF), 其推测的氨基酸序列具有八氢番茄红素脱氢酶的典型结构域。RhPDS1基因在蕾、花和嫩叶中的相对表达量较高。利用基因编辑获得3个具有白化表型的再生株系, 白化苗分化率为3.7%。测序结果表明, 3个突变体属于2个基因编辑事件, 靶点序列分别为1 bp或/和5 bp碱基缺失, 造成移码突变。白化苗突变体的叶绿素和类胡萝卜素含量极显著低于野生型, RhPDS1基因的表达量也显著下降。综上, 该研究克隆了RhPDS1基因, 并利用CRISPR/Cas9技术实现了对RhPDS1基因的靶向敲除, 为湖北地黄的功能基因组学研究和野生驯化奠定了技术基础。
苗春妍, 李铭铭, 左鑫, 丁宁, 杜家方, 李娟, 张重义, 王丰青. 湖北地黄CRISPR/Cas9基因编辑体系的建立. 植物学报, 2023, 58(6): 905-916.
Chunyan Miao, Mingming Li, Xin Zuo, Ning Ding, Jiafang Du, Juan Li, Zhongyi Zhang, Fengqing Wang. Establishment of CRISPR/Cas9 Gene Editing System in Rehmannia henryi. Chinese Bulletin of Botany, 2023, 58(6): 905-916.
Primer name | Primer sequence (5′-3′) | Aim |
---|---|---|
RhPDS1_orf_F | CCAAAAGTTGCTCCACCTGT | Amplification of RhPDS1 from cDNA |
RhPDS1_orf_R | TGCTGCTTCCCTATCTTCTG | |
RhPDS1_F | ATATCAAAATGGCCCAATTCGGA | Amplification of RhPDS1 from genomic DNA |
RhPDS1_R | TCCACACTCGAAACATATTAGGC | |
RhPDS1q_F | GCTAGCAGATGTGGCAGC | qRT-PCR analysis of RhPDS1 |
RhPDS1q_R | CAATGACCACTTGCAATGGC | |
RhActin_F | GTGAGGAGATGCAGAAGCAA | qRT-PCR analysis of RhActin |
RhActin_R | CCTCGTGTGTTCACTGACAA | |
Rh18S_F | AGCAAGGGCAGTGTTACAAG | qRT-PCR analysis of Rh18S |
Rh18S_R | GACCACAAGTGAGGACAAGG | |
sgRhPDS1_F | attgAAGCAAGGGATGTGCTGGG | Construction of target site |
sgRhPDS1_R | aaacCCCAGCACATCCCTTGCTT | |
Detect1_F | TCAACGGCATTCGGGACAAG | Identification of the transformation |
Detect1_R | CCACATACATATCGCGGCCA | |
Detect2_F | TGTCCCAGGATTAGAATGATTAGGC | Identification of the transformation |
Detect2_R | CCCAGCACATCCCTTGCTT | |
RhPDS1t_F | CGACTATCCGAGACCAGAGC | Amplification of target region |
RhPDS1t_R | ATGTGCAACCCAGTCTCGTA |
表1 引物名称及序列
Table 1 Primer names and sequences
Primer name | Primer sequence (5′-3′) | Aim |
---|---|---|
RhPDS1_orf_F | CCAAAAGTTGCTCCACCTGT | Amplification of RhPDS1 from cDNA |
RhPDS1_orf_R | TGCTGCTTCCCTATCTTCTG | |
RhPDS1_F | ATATCAAAATGGCCCAATTCGGA | Amplification of RhPDS1 from genomic DNA |
RhPDS1_R | TCCACACTCGAAACATATTAGGC | |
RhPDS1q_F | GCTAGCAGATGTGGCAGC | qRT-PCR analysis of RhPDS1 |
RhPDS1q_R | CAATGACCACTTGCAATGGC | |
RhActin_F | GTGAGGAGATGCAGAAGCAA | qRT-PCR analysis of RhActin |
RhActin_R | CCTCGTGTGTTCACTGACAA | |
Rh18S_F | AGCAAGGGCAGTGTTACAAG | qRT-PCR analysis of Rh18S |
Rh18S_R | GACCACAAGTGAGGACAAGG | |
sgRhPDS1_F | attgAAGCAAGGGATGTGCTGGG | Construction of target site |
sgRhPDS1_R | aaacCCCAGCACATCCCTTGCTT | |
Detect1_F | TCAACGGCATTCGGGACAAG | Identification of the transformation |
Detect1_R | CCACATACATATCGCGGCCA | |
Detect2_F | TGTCCCAGGATTAGAATGATTAGGC | Identification of the transformation |
Detect2_R | CCCAGCACATCCCTTGCTT | |
RhPDS1t_F | CGACTATCCGAGACCAGAGC | Amplification of target region |
RhPDS1t_R | ATGTGCAACCCAGTCTCGTA |
图1 RhPDS1基因的PCR扩增产物电泳检测 (A) 以湖北地黄叶片cDNA为模板扩增RhPDS1; (B) 以湖北地黄叶片基因组DNA为模板扩增RhPDS1基因
Figure 1 Electrophoresis detection of PCR amplified products of RhPDS1 gene (A) RhPDS1 was amplified with the cDNA of Rehmannia henryi leaves as a template; (B) RhPDS1 was amplified with the genomic DNA of R. henryi leaves as a template
图2 RhPDS1的序列特征及基因表达特性 (A) RhPDS1与拟南芥、地黄和天目地黄同源蛋白的多序列联配; (B) RhPDS1及其同源蛋白的系统进化树; (C) 湖北地黄RhPDS1基因的组织表达特性(不同小写字母表示不同组织间基因表达差异显著(P<0.05))。
Figure 2 Sequence characteristics and gene expression patterns of RhPDS1 (A) Multiple sequence alignment of RhPDS1 with its homologs from Arabidopsis thaliana, Rehmannia glutinosa and R. chingii; (B) Phylogenetic tree of RhPDS1 and its homologous proteins; (C) Expression profiles of RhPDS1 in different tissues of R. henryi (different lowercase letters represent significant differences among different tissues (P<0.05)).
图3 RhPDS1基因编辑载体构建及湖北地黄遗传转化 (A) RhPDS1基因编辑载体框架图; (B) 抗性愈伤组织筛选(bar=1 cm); (C), (D) 抗性芽分化(bars=1 cm); (E) 白化芽再生(bar=1 cm); (F) 白化苗的PCR鉴定。PAM: 原间隔序列邻近基序; WT: 野生型
Figure 3 Construction of genome editing vector and genetic transformation of Rehmannia henryi (A) Schematic map of CRISPR/Cas9 construct for RhPDS1; (B) Embryogenic calli were screened on the medium with kanamycine (bar=1 cm); (C), (D) Resistant shoots differentiation (bars=1 cm); (E) Albino shoot regeneration (bar=1 cm); (F) Positive identification of albino shoots by PCR. PAM: Protospacer adjacent motif; WT: Wild type
图4 转基因湖北地黄RhPDS1基因靶点突变分析 (A) 3株白化苗的表型(bars=1 cm); (B) 2种白化突变体的靶点基因组碱基序列特征(短横线代表缺失碱基); (C) 2种突变的T克隆测序峰图; (D) 2种突变体的氨基酸序列特征。PAM和WT同表3。
Figure 4 Analysis of target mutations of RhPDS1 gene in transgenic Rehmannia henryi (A) Phenotypes of the three albino shoots (bars=1 cm); (B) cDNA sequence characteristics of target site in the genome of two albino mutants (short horizontal lines represent base deletions); (C) Sanger sequencing results for T clone of two kinds of mutant; (D) Amino acids sequence characteristics of the two albino mutants. PAM and WT are the same as shown in Figure 3.
图5 基因编辑突变体的叶绿素、类胡萝卜素含量及RhPDS1的表达量 (A) 突变体的叶绿素和类胡萝卜素含量; (B) 突变体中RhPDS1的相对表达量。数据为3次生物学重复平均值±标准差(n=3)。 **表示与野生型(WT)相比在P<0.01水平差异显著。
Figure 5 Detection of chlorophylls and carotenoids contents in RhPDS1 mutants and the expression of RhPDS1 (A) Chlorophylls and carotenoids contents in RhPDS1 mutants; (B) The relative expression levels of RhPDS1 in CRISPR/Cas9 edited shoots. Values are means±SD (n=3). ** indicate significant differences compared to wild type (WT) at P<0.01.
[1] |
郭丽, 王雪涵, 田丰 (2023). 多组学整合网络: 一把精准解码玉米功能基因组的钥匙. 植物学报 58, 1-5.
DOI |
[2] |
何晓玲, 刘鹏程, 马伯军, 陈析丰 (2022). 基于CRISPR/ Cas9的基因编辑技术研究进展及其在植物中的应用. 植物学报 57, 508-531.
DOI |
[3] | 彭华胜, 徐长青, 袁媛, 查良平, 陈焕文, 管理, 康利平, 杨军, 王亚君, 曹丽娟, 程京, 黄璐琦 (2019). 最早的中药辅料炮制品: 西汉海昏侯墓出土的木质漆盒内样品鉴定与分析. 科学通报 64, 935-947. |
[4] |
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, 791-800.
DOI |
[5] | 吴琼, 孙超, 陈士林, 罗红梅, 李滢, 孙永珍, 牛云云 (2010). 转录组学在药用植物研究中的应用. 世界科学技术(中医药现代化) 12, 457-462. |
[6] | 周婕 (2019). 湖北地黄化学成分研究. 硕士论文. 北京: 北京协和医学院. pp. 32-102. |
[7] |
左鑫, 李铭铭, 李欣容, 苗春妍, 李炎枋, 杨旭, 张重义, 王丰青 (2022). CRISPR/Cas9技术在天目地黄RcPDS1基因编辑中的应用. 园艺学报 49, 1532-1544.
DOI |
[8] |
Alagoz Y, Gurkok T, Zhang BH, 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. Sci Rep 6, 30910.
DOI PMID |
[9] |
Banakar R, Schubert M, Collingwood M, Vakulskas C, Eggenberger AL, Wang K (2020). Comparison of CRISPR-Cas9/Cas12a ribonucleoprotein complexes for genome editing efficiency in the rice phytoene desaturase (OsPDS) gene. Rice 13, 4.
DOI PMID |
[10] |
Bánfalvi Z, Csákvári E, Villányi V, Kondrák M (2020). Generation of transgene-free PDS mutants in potato by Agrobacterium-mediated transformation. BMC Biotechnol 20, 25.
DOI PMID |
[11] |
Cheng JY, Hill C, Han Y, He TH, Ye XG, Shabala S, Guo GG, Zhou MX, Wang K, Li CD (2023). New semi-dwarfing alleles with increased coleoptile length by gene editing of gibberellin 3-oxidase 1 using CRISPR-Cas9 in barley (Hordeum vulgare L.). Plant Biotechnol J 21, 806-818.
DOI URL |
[12] |
Kaur N, Alok A, Shivani, Kaur N, Pandey P, Awasthi P, Tiwari S (2018). CRISPR/Cas9-mediated efficient editing in phytoene desaturase (PDS) demonstrates precise manipulation in banana cv. Rasthali genome. Funct Integr Genomics 18, 89-99.
DOI URL |
[13] |
Koschmieder J, Fehling-Kaschek M, Schaub P, Ghisla S, Brausemann A, Timmer J, Beyer P (2017). Plant-type phytoene desaturase: functional evaluation of structural implications. PLoS One 12, e0187628.
DOI URL |
[14] | Kui L, Chen HT, Zhang WX, He SM, Xiong ZJ, Zhang YS, Yan L, Zhong CF, He FM, Chen JW, Zeng P, Zhang GH, Yang SC, 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. Front Plant Sci 7, 2036. |
[15] |
Li TD, Yang XP, Yu Y, Si XM, Zhai XW, Zhang HW, Dong WX, Gao CX, Xu C (2018). Domestication of wild tomato is accelerated by genome editing. Nat Biotechnol 36, 1160-1163.
DOI |
[16] |
Li XR, Zuo X, Li MM, Yang X, Zhi JY, Sun HZ, Xie CX, Zhang ZY, Wang FQ (2021). Efficient CRISPR/Cas9- mediated genome editing in Rehmannia glutinosa. Plant Cell Rep 40, 1695-1707.
DOI |
[17] | Ma CF, Liu MC, Li QF, Si J, Ren XS, Song HY (2019). Efficient BoPDS gene editing in cabbage by the CRISPR/Cas9 system. Hortic Plant J 5, 164-169. |
[18] |
Ma LG, Dong CM, Song C, Wang XL, Zheng XK, Niu Y, Chen SL, Feng WS (2021). De novo genome assembly of the potent medicinal plant Rehmannia glutinosa using nanopore technology. Comput Struct Biotechnol J 19, 3954-3963.
DOI URL |
[19] |
Mao YF, Zhang ZJ, Feng ZY, Wei PL, Zhang H, Botella JR, Zhu JK (2016). Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis. Plant Biotechnol J 14, 519-532.
DOI URL |
[20] |
Odipio J, Alicai T, Ingelbrecht I, Nusinow DA, Bart R, Taylor NJ (2017). Efficient CRISPR/Cas9 genome editing of Phytoene desaturase in cassava. Front Plant Sci 8, 1780.
DOI PMID |
[21] |
Qin GJ, Gu HY, Ma LG, Peng YB, Deng XW, Chen ZL, Qu LJ (2007). Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis. Cell Res 17, 471-482.
DOI |
[22] |
Ren C, Liu YF, Guo YC, Duan W, Fan PG, Li SH, Liang ZC (2021). Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters. Hortic Res 8, 52.
DOI |
[23] |
Schmittgen TD, Livak KJ (2008). Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3, 1101-1108.
DOI PMID |
[24] |
Son S, Park SR (2022). Challenges facing CRISPR/Cas9- based genome editing in plants. Front Plant Sci 13, 902413.
DOI URL |
[25] |
Wang FQ, Li XR, Zuo X, Li MM, Miao CY, Zhi JY, Li YJ, Yang X, Liu XY, Xie CX (2021). Transcriptome-wide identification of WRKY transcription factor and functional characterization of RgWRKY37 involved in acteoside biosynthesis in Rehmannia glutinosa. Front Plant Sci 12, 739853.
DOI URL |
[26] |
Wang J, Wu HT, Chen YN, Yin TM (2020). Efficient CRISPR/Cas9-mediated gene editing in an interspecific hybrid poplar with a highly heterozygous genome. Front Plant Sci 11, 996.
DOI PMID |
[27] |
Wilson FM, Harrison K, Armitage AD, Simkin AJ, Harrison RJ (2019). CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in diploid and octoploid strawberry. Plant Methods 15, 45.
DOI PMID |
[28] |
Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ (2014). A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14, 327.
DOI URL |
[29] |
Xu ZS, Feng K, Xiong AS (2019). CRISPR/Cas9-mediated multiply targeted mutagenesis in orange and purple carrot plants. Mol Biotechnol 61, 191-199.
DOI |
[30] |
Yu H, Lin T, Meng XB, Du HL, Zhang JK, Liu GF, Chen MJ, Jing YH, Kou LQ, Li XX, Gao Q, Liang Y, Liu XD, Fan ZL, Liang YT, Cheng ZK, Chen MS, Tian ZX, Wang YH, Chu CC, Zuo JR, Wan JM, Qian Q, Han B, Zuccolo A, Wing RA, Gao CX, Liang CZ, Li JY (2021). A route to de novo domestication of wild allotetraploid rice. Cell 184, 1156-1170.
DOI URL |
[31] |
Zhang YX, Malzahn AA, Sretenovic S, Qi YP (2019). The emerging and uncultivated potential of CRISPR technology in plant science. Nat Plants 5, 778-794.
DOI PMID |
[32] |
Zhang YX, Xu GC, Cheng CH, Lei L, Sun J, Xu Y, Deng CH, Dai ZG, Yang ZM, Chen XJ, Liu C, Tang Q, Su JG (2021). Establishment of an Agrobacterium-mediated genetic transformation and CRISPR/Cas9-mediated targeted mutagenesis in hemp (Cannabis sativa L.). Plant Biotechnol J 19, 1979-1987.
DOI URL |
[33] |
Zhou J, Shi GR, Liu YF, Chen RY, Yu DQ (2019). Nine new compounds from the whole plants of Rehmannia henryi. J Asian Nat Prod Res 21, 399-408.
DOI PMID |
[34] |
Zhou Z, Tan HX, Li Q, Chen JF, Gao SH, Wang Y, Chen WS, Zhang L (2018). CRISPR/Cas9-mediated efficient targeted mutagenesis of RAS in Salvia miltiorrhiza. Phytochemistry 148, 63-70.
DOI URL |
[35] |
Zsögön A, Čermák T, Naves ER, Notini MM, Edel KH, Weinl S, Freschi L, Voytas DF, Kudla J, Peres LEP (2018). De novo domestication of wild tomato using genome editing. Nat Biotechnol 36, 1211-1216.
DOI |
[36] | Zuo X, Wang FQ, Li XR, Li MM (2020). Transcriptome- based screening and the optimal reference genes for real-time quantitative PCR in Rehmannia chingii and R. henryi. Biol Plant 64, 798-806. |
[1] | 何晓玲, 刘鹏程, 马伯军, 陈析丰. 基于CRISPR/Cas9的基因编辑技术研究进展及其在植物中的应用[J]. 植物学报, 2022, 57(4): 508-531. |
[2] | 谢先荣, 曾栋昌, 谭健韬, 祝钦泷, 刘耀光. 基于CRISPR编辑系统的DNA片段删除技术[J]. 植物学报, 2021, 56(1): 44-49. |
[3] | 苏钺凯,邱镜仁,张晗,宋振巧,王建华. CRISPR/Cas9系统在植物基因组编辑中技术改进与创新的研究进展[J]. 植物学报, 2019, 54(3): 385-395. |
[4] | 王影, 李相敢, 邱丽娟. CRISPR/Cas9基因组定点编辑中脱靶现象的研究进展[J]. 植物学报, 2018, 53(4): 528-541. |
阅读次数 | ||||||
全文 |
|
|||||
摘要 |
|
|||||