植物学报 ›› 2023, Vol. 58 ›› Issue (2): 298-307.DOI: 10.11983/CBB22240
邱锐1,2, 何峰2, 李瑞2, 王亚梅2, 邢思年2, 曹英萍2, 刘叶飞1,2, 周昕越1, 赵彦1,*(), 付春祥2,*(
)
收稿日期:
2022-10-13
接受日期:
2023-01-16
出版日期:
2023-03-01
发布日期:
2023-03-15
通讯作者:
*E-mail: 基金资助:
Rui Qiu1,2, Feng He2, Rui Li2, Yamei Wang2, Sinian Xing2, Yingping Cao2, Yefei Liu1,2, Xinyue Zhou1, Yan Zhao1,*(), Chunxiang Fu2,*(
)
Received:
2022-10-13
Accepted:
2023-01-16
Online:
2023-03-01
Published:
2023-03-15
Contact:
*E-mail: 摘要: 柳枝稷(Panicum virgatum)是重要的C4多年生木质纤维素类生态能饲草。为了快速创制细胞壁转化效率高的能饲草新资源, 以异源四倍体柳枝稷品种Alamo为材料, 克隆了其木质素合成途径的阿魏酸-5-羟基化酶基因PvF5H, 并根据其序列设计编辑靶点, 用于构建CRISPR/Cas9-PvF5H编辑载体, 最后通过农杆菌(Agrobacterium tumefaciens)介导的遗传转化方法, 获得了59株柳枝稷转基因阳性植株。测序分析表明, PvF5H在94.9%的转基因植株中被编辑, 纯合编辑效率为55.4%。该研究建立了高效的柳枝稷基因编辑系统, 实现了对细胞壁品质相关靶基因的有效编辑, 为今后能饲草新品种的培育奠定了基础。
邱锐, 何峰, 李瑞, 王亚梅, 邢思年, 曹英萍, 刘叶飞, 周昕越, 赵彦, 付春祥. 柳枝稷木质素基因F5H的高效编辑. 植物学报, 2023, 58(2): 298-307.
Rui Qiu, Feng He, Rui Li, Yamei Wang, Sinian Xing, Yingping Cao, Yefei Liu, Xinyue Zhou, Yan Zhao, Chunxiang Fu. Highly Efficient Gene Editing of Lignin Gene F5H in Switchgrass. Chinese Bulletin of Botany, 2023, 58(2): 298-307.
图1 柳枝稷PvF5H的基本分子特征 (A) 柳枝稷F5H基因的染色体定位(PvF5Ha: Pavir.9NG241700.1; PvF5Hb: Pavir.9KG138400.1); (B) 柳枝稷F5H基因的外显子与内含子分布图(蓝色方框为外显子, 黑色细线为内含子, 黑色粗线为5'和3'非翻译序列); (C) 不同物种中F5H蛋白的系统进化树及基因保守基序分析(柳枝稷: Pavir.9NG241700.1 (PvF5Ha)、Pavir.9KG138400.1 (PvF5Hb); 水稻: LOC_Os10g36848.1 (OsF5H); 玉米: ZmPHB47.01G353500.1 (ZmF5H); 高粱: Sobic.001G196300.1.p (SsF5H); 蒺藜苜蓿: Medtr5g021390.1 (Mt- F5H); 拟南芥: AT4G36220.1 (AtF5H)。基序分析中不同颜色元件代表不同核苷酸序列)
Figure 1 The basic molecular characterization of PvF5H in Panicum virgatum (A) Chromosomal position of F5H gene in P. virgatum (PvF5Ha: Pavir.9NG241700.1; PvF5Hb: Pavir.9KG138400.1); (B) F5H gene exons and introns distribution map of P. virgatum (blue boxes are exons, black thin lines are introns, thick black lines are 5' and 3' untranslated sequences); (C) Phylogenetic tree of F5H protein and gene conserved motif analysis in different species (P. virgatum: Pavir.9NG241700.1 (PvF5Ha), Pavir.9KG138400.1 (PvF5Hb); Oryza sativa: LOC_Os10g36848.1 (OsF5H); Zea mays: ZmPHB47.01G353500.1 (ZmF5H); Sorghum bicolor: Sobic.001G196300.1.p (SsF5H); Medicago truncatula: Medtr5g- 021390.1 (MtF5H); Arabidopsis thaliana: AT4G362201.1 (AtF5H). Different color of elements represent different nucleotide sequences in motif analysis)
图2 柳枝稷PvF5H基因编辑载体的构建 (A) PvF5H1a和PvF5H1b基因序列比对(红框表示靶位点); (B) 柳枝稷F5H基因编辑位点示意图(蓝色代表外显子, 细线代表内含子); (C) 柳枝稷F5H gRNA靶点+gRNA骨架的二级结构预测图; (D) F5H基因编辑载体图谱(该载体通过玉米Ubiquitin基因启动子启动Cas9表达, OsU3启动sgRNA表达)
Figure 2 Construction of PvF5H gene editing vector in Panicum virgatum (A) PvF5H1a and PvF5H1b gene sequence alignment (red box indicates target site); (B) Schematic diagram of F5H gene editing site in switchgrass (blue for exons, thin lines for introns); (C) Secondary structure prediction map of F5H gRNA target+gRNA skeleton in switchgrass; (D) F5H gene editing vector map (the expression of Cas9 was driven by maize Ubiquitin promoter and sgRNA by OsU3 promoter)
图3 CRISPR/Cas9_PvF5H转基因柳枝稷的产生与鉴定 (A) 柳枝稷愈伤组织在培养基上进行抗性筛选; (B) 抗性筛选后的愈伤组织在分化培养基上变绿并且产生不定芽; (C) 不定芽伸长; (D) 伸长的不定芽在生根培养基中诱导生根; (E) 柳枝稷转基因植株的PCR鉴定。阴性对照(-, 野生型柳枝稷)均未扩增出目的基因(hph和Cas9)片段条带, 而转基因阳性植株(2-20泳道)均扩增出与阳性对照(+, CRISPR/Cas9_PvF5H载体质粒)相同大小的条带。(A)-(D) Bars=1 cm
Figure 3 Generation and identification of CRISPR/Cas9_PvF5H transgenic switchgrass (A) Screening of switchgrass resistant calli grown on medium; (B) Calli turned green and produced shoots on differentiation medium after resistance screening; (C) Shoot enlongation; (D) Roots formation in rooting medium; (E) PCR analysis of transgenic switchgrass lines. No fragment band was amplified in the negative control (-, wild type switchgrass), while the same-sized band were amplified in all the resistant plants (lane 2 to 20) as in the positive control (+, CRISPR/Cas9_PvF5H plasmid). (A)-(D) Bars=1 cm
图4 柳枝稷基因编辑方法及效率 (A) 柳枝稷F5H基因编辑的效率; (B) 柳枝稷PvF5H基因的编辑模式(I: 插入; D: 删除; D&I: 删除+插入)
Figure 4 Gene editing methods and efficiency in switchgrass (A) Efficiency of F5H gene editing in switchgrass; (B) Gene editing patterns of PvF5H in switchgrass (I: Insertion; D: Delete; D&I: Delete+insertion)
[1] |
何海锋, 吴娜, 刘吉利, 陈娟, 刘晓侠, 常雯雯 (2020). 柳枝稷种植年限对盐碱土壤理化性质的影响. 生态环境学报 29, 285-292.
DOI |
[2] | 金枝, 陈倩, 代琳心, 马建锋 (2022). 木质纤维细胞壁大分子取向研究进展. 北京林业大学学报 44(12), 153-160. |
[3] |
林萌萌, 李春娟, 闫彩霞, 孙全喜, 赵小波, 王娟, 苑翠玲, 单世华 (2021). CRISPR/Cas9基因编辑技术在作物中的应用. 核农学报 35, 1329-1339.
DOI |
[4] | 刘吉利, 朱万斌, 谢光辉, 林长松, 程序 (2009). 能源作物柳枝稷研究进展. 草业学报 18(3), 232-240. |
[5] | 马永清, 郝智强, 熊韶峻, 刘吉利 (2012). 我国柳枝稷规模化种植现状与前景. 中国农业大学学报 17(6), 133-137. |
[6] | 王进 (2009). 亚麻(Linum usitatissimum)木质素合成关键酶基因的克隆及表达分析. 硕士论文. 北京: 中国农业科学院. pp. 3-5. |
[7] | 徐超, 方志, 杨芳梅, 杨君祎, 闫冲冲, 邹鹤伟, 张金云, 林毅, 蔡永萍 (2015). 砀山酥梨果实F5H表达与石细胞发育的分析. 植物生理学报 51, 778-784. |
[8] |
Akbas MY, Stark BC (2016). Recent trends in bioethanol production from food processing byproducts. J Ind Microbiol Biotechnol 43, 1593-1609.
DOI URL |
[9] |
Amthor JS (2003). Efficiency of lignin biosynthesis: a quantitative analysis. Ann Bot 91, 673-695.
DOI URL |
[10] |
Andersen JR, Zein I, Wenzel G, Darnhofer B, Eder J, Ouzunova M, Lübberstedt T (2008). Characterization of phenylpropanoid pathway genes within European maize (Zea mays L.) inbreds. BMC Plant Biol 8, 2.
DOI PMID |
[11] |
Baskin TI (2017). Plant cell growth: cellulose caught slipping. Nat Plants 3, 17063.
DOI PMID |
[12] | Baxter HL, Mazarei M, Labbe N, Kline LM, Cheng QK, Windham MT, Mann DGJ, Fu CX, Ziebell A, Sykes RW, Rodriguez M Jr, Davis MF, Mielenz JR, Dixon RA, Wang ZY, Stewart CN Jr (2014). Two-year field analysis of reduced recalcitrance transgenic switchgrass. Plant Bio- technol J 12, 914-924. |
[13] |
Boerjan W, Ralph J, Baucher M (2003). Lignin biosynthesis. Annu Rev Plant Biol 54, 519-546.
PMID |
[14] |
Bortesi L, Fischer R (2015). The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33, 41-52.
DOI PMID |
[15] |
Chen CJ, Chen H, Zhang Y, Thomas HR, Frank MH, He YH, Xia R (2020). TBtools: an integrative toolkit deveoped for interactive analyses of big biological data. Mol Plant 13, 1194-1202.
DOI URL |
[16] |
Chen F, Dixon RA (2007). Lignin modification improves fermentable sugar yields for biofuel production. Nat Biotechnol 25, 759-761.
DOI PMID |
[17] |
Fu CX, Mielenz JR, Xiao XR, Ge YX, Hamilton CY, Rodriguez M Jr, Chen F, Foston M, Ragauskas A, Bouton J, Dixon RA, Wang ZY (2011). Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass. Proc Natl Acad Sci USA 108, 3803-3808.
DOI PMID |
[18] |
Guo D, Chen F, Inoue K, Blount JW, Dixon RA (2001). Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa. impacts on lignin structure and implications for the biosynthesis of G and S lignin. Plant Cell 13, 73-88.
DOI PMID |
[19] |
Humphreys JM, Chapple C (2002). Rewriting the lignin roadmap. Curr Opin Plant Biol 5, 224-229.
DOI PMID |
[20] | Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Scien- ce 337, 816-821. |
[21] |
Jung JH, Fouad WM, Vermerris W, Gallo M, Altpeter F (2012). RNAi suppression of lignin biosynthesis in sugarcane reduces recalcitrance for biofuel production from lignocellulosic biomass. Plant Biotechnol J 10, 1067-1076.
DOI PMID |
[22] |
Keshwani DR, Cheng JJ (2009). Switchgrass for bioethanol and other value-added applications: a review. Bioresour Technol 100, 1515-1523.
DOI URL |
[23] | Kim J, Choi B, Park YH, Cho BK, Lim HS, Natarajan S, Park SU, Bae H (2013). Molecular characterization of ferulate 5-hydroxylase gene from kenaf (Hibiscus cannabinus L.). Sci World J 2013, 421578. |
[24] |
Lapierre C, Pollet B, Petit-Conil M, Toval G, Romero J, Pilate G, Leplé JC, Boerjan W, Ferret V, De Nadai V, Jouanin L (1999). Structural alterations of lignins in transgenic poplars with depressed cinnamyl alcohol dehydrogenase or caffeic acid O-methyltransferase activity have an opposite impact on the efficiency of industrial kraft pulping. Plant Physiol 119, 153-164.
DOI PMID |
[25] | Lovell JT, MacQueen AH, Mamidi S, Bonnette J, Jenkins J, Napier JD, Sreedasyam A, Healey A, Session A, Shu SQ, Barry K, Bonos S, Boston L, Daum C, Deshpande S, Ewing A, Grabowski PP, Haque T, Harrison M, Jiang JM, Kudrna D, Lipzen A, Pendergast IV TH, Plott C, Qi P, Saski CA, Shakirov EV, Sims D, Sharma M, Sharma R, Stewart A, Singan VR, Tang YH, Thibivillier S, Webber J, Weng XY, Williams M, Wu GA, Yoshinaga Y, Zane M, Zhang L, Zhang JY, Behrman KD, Boe AR, Fay PA, Fritschi FB, Jastrow JD, Lloyd-Reilley J, Martínez-Reyna JM, Matamala R, Mitchell RB, Rouquette FM Jr, Ronald P, Saha M, Tobias CM, Udvardi M, Wing RA, Wu YQ, Bartley LE, Casler M, Devos KM, Lowry DB, Rokhsar DS, Grimwood J, Juenger TE, Schmutz J (2021). Genomic mechanisms of climate adaptation in polyploid bioenergy switchgrass. Nature 590, 438-444. |
[26] |
Meyer K, Cusumano JC, Somerville C, Chapple CC (1996). Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monoxygenases. Proc Natl Acad Sci USA 93, 6869-6874.
PMID |
[27] | Nageswara-Rao M, Soneji JR, Kwit C, Stewart CN Jr (2013). Advances in biotechnology and genomics of swi- tchgrass. Biotechnol Biofuels 6, 77. |
[28] |
Park JJ, Yoo CG, Flanagan A, Pu YQ, Debnath S, Ge YX, Ragauskas AJ, Wang ZY (2017). Defined tetra-allelic gene disruption of the 4-coumarate: coenzyme A ligase 1 (Pv4CL1) gene by CRISPR/Cas9 in switchgrass results in lignin reduction and improved sugar release. Biotechnol Biofuels 10, 284.
DOI |
[29] |
Raes J, Rohde A, Christensen JH, Van de Peer Y, Boerjan W (2003). Genome-wide characterization of the lignification toolbox in Arabidopsis. Plant Physiol 133, 1051-1071.
DOI URL |
[30] |
Sakiroglu M, Sherman-Broyles S, Story A, Moore KJ, Doyle JJ, Brummer EC (2012). Patterns of linkage dise- quilibrium and association mapping in diploid alfalfa (M. sativa L.). Theor Appl Genet 125, 577-590.
DOI PMID |
[31] |
Shen H, He XZ, Poovaiah CR, Wuddineh WA, Ma JY, Mann DGJ, Wang HZ, Jackson L, Tang YH, Neal Stewart C Jr, Chen F, Dixon RA (2012). Functional characterization of the switchgrass (Panicum virgatum) R2R3-MYB transcription factor PvMYB4 for improvement of lignocellulosic feedstocks. New Phytol 193, 121-136.
DOI PMID |
[32] | Shen H, Mazarei M, Hisano H, Escamilla-Trevino L, Fu CX, Pu YQ, Rudis MR, Tang YH, Xiao XR, Jackson L, Li GF, Hernandez T, Chen F, Ragauskas AJ, Stewart CN Jr, Wang ZY, Dixon RA (2013). A genomics approach to deciphering lignin biosynthesis in switchgrass. Plant Cell 25, 4342-4361. |
[33] | Stewart JJ, Akiyama T, Chapple C, Ralph J, Mansfield SD (2009). The effects on lignin structure of overexpression of ferulate 5-hydroxylase in hybrid poplar. Plant Phy- siol 150, 621-635. |
[34] | Wu ZY, Wang NF, Hisano H, Cao YP, Wu FY, Liu WW, Bao Y, Wang ZY, Fu CX (2019). Simultaneous regulation of F5H in COMT-RNAi transgenic switchgrass alterse- fects of COMT suppression on syringyl lignin biosynthe-sis. Plant Biotechnol J 17, 836-845. |
[35] |
Xu B, Escamilla-Treviño LL, Sathitsuksanoh N, Shen ZX, Shen H, Zhang YHP, Dixon RA, Zhao BY (2011). Silencing of 4-coumarate: coenzyme A ligase in switchgrass leads to reduced lignin content and improved fermentable sugar yields for biofuel production. New Phytol 192, 611-625.
DOI URL |
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