Creating Rice Gerplasm Resources OsALS Rsistant to Herbicide through Single Base Gene Editing Technology

doi:10.13560/j.cnki.biotech.bull.1985.2024-0780

Abstract

Abstract:

Objective This study edited the acetyl lactate synthase (ALS) gene at designated locus of Ningjing 56, Ningxia salt-tolerant variety, and created rice germplasm resources with herbicide resistance characteristics. Method Using the cytosine base editor BE3, single base editing vector OsALS171-SpG-eBE3 and OsALS190-SpG-eBE3 were constructed with ALS gene 171 and 190 as target sites. Gene edited plants were obtained through Agrobacterium mediated transformation method and target sites were genotyped with Sanger sequencing. A rapid herbicide tolerance system was established and tolerance level was identified in edited herbicide materials. Result Through T₁ target sites sequencing, Two ALS^P171Fmutant homozygous lines and one homozygous strain of ALS^{E187K R190H}, ALS^E187K, ALS^V188I, ALS^R190H, and ALS^D201Nmutation types were obtained. Compared to wild-type material, Two ALS^P171F mutant homozygous strains have strong tolerance to bispyribac-sodium(BS), while ALS^{E187K R190H}, ALS^E187K, ALS^V188I, ALS^R190H, and ALS^D201Nmutant homozygous plants have some tolerances to BS, but the tolerance is not significant. Conclusion The gene edited materials can maintain stable inheritance, using the cytosine base editor BE3 to obtain materials with herbicide resistance characteristics, and the ALS^P171Fmutation demonstrates strong resistance to bispyribac-sodium, which is a priority site for cultivating herbicide resistant rice.

Key words: rice, single base gene editing, herbicide-resistant, acetolactate synthase

CHEN Xiao-jun, HUI Jian, MA Hong-wen, BAI Hai-Bo, ZHONG Nan, LI Jia-run, FAN Yun-fang. Creating Rice Gerplasm Resources OsALS Rsistant to Herbicide through Single Base Gene Editing Technology[J]. Biotechnology Bulletin, 2025, 41(4): 106-114.

Figures/Tables 10

Fig. 1 Gene structure of OsALS and edited targets in OsALSThe red letters indicate PAM structure

Table 1 List of designed primers

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	用途 Purpose
OsALS171-gRNA-F	ggcaGTCCCCCGCCGCATGAT	OsALS171位点基因编辑载体构建
OsALS171-gRNA-R	aaacATCATGCGGCGGGGGAC	OsALS171位点基因编辑载体构建
OsALS190-gRNA-F	ggcaGGAGCGGGTGACCTCGACTA	OsALS190位点基因编辑载体构建
OsALS190-gRNA-R	aaacTAGTCGAGGTCACCCGCTCC	OsALS190位点基因编辑载体构建
OsALS-dect-F	GCTCGGCGGTGTCCCCGGTC	两个靶位点检测
OsALS-dect-R	CCGGCACGGCCATCTGCTGC	两个靶位点检测

Fig. 2 Schematic diagram of SpG-eBE3 plant single base gene editing vector

Fig. 3 Sequencing of gRNA in plant single base editing vectorA: Sequencing of target sites in vector OsALS171-SpG-eBE3. B: Sequencing of target sites in vector OsALS190-SpG-eBE3

Fig. 4 Editing results of OsALS171-SpG-eBE3 in T0 plantsThe overlapping peak of cytosine base C and thymine base T indicates edited bases in target sites

Fig. 5 Editing results of OsALS190-SpG-eBE3 in T0 plantsThe overlapping peak indicates edited bases in target sites

Fig. 6 Editing results of OsALS171-SpG-eBE3 in T1 plantsP amino acids mutated F amino acids at 171 of OsALS in D12-9 and D13-4 plants

Fig. 7 Editing results of OsALS171-SpG-eBE3 in T1 plantsE amino acids mutated K amino acids at 187 of OsALS and R amino acids mutated H amino acids at 190 of OsALS in D15-25 plants. E amino acids mutated K amino acids at 187 of OsALS in D16-6 plants. V amino acids mutated I amino acids at 188 of OsALS in D40-24 plants. R amino acids mutated H amino acids at 190 of OsALS in D24-1 plants. D amino acids mutated N amino acids at 201 of OsALS in D57-10 plants

Fig. 8 Characterization of wild-type receptor material for resistance to bispyribac-sodium

Fig. 9 Characterization of edited homozygote for resistance to bispyribac-sodiumA: Wild type receptor plants; B: D12-9 homozygote plants; C: D13-4 homozygote plants; D: D15-25 homozygote plants; E: D16-6 homozygote plants; F: D40-24 homozygote plants; G: D24-1 homozygote plants; H: D57-10 homozygote plants. In each group, the left-to-right bispyribac-sodium concentrations are 0, 0.2, and 0.4 μmol/L, respectively

References 37

1	曾波. 近30年来我国水稻主要品种更新换代历程浅析 [J]. 作物杂志, 2018(3): 1-7.
	Zeng B. Renovation of main cultivated rice varieties in China in the past 30 years [J]. Crops, 2018(3): 1-7.
2	黄健, 朱安, 汪浩, 等. 水直播和旱直播对水稻产量与品质的影响综述 [J]. 江苏农业科学, 2020, 48(16): 67-73.
	Huang J, Zhu A, Wang H, et al. Effects of water direct-seeding and dry direct-seeding on yield and quality of rice: a review [J]. Jiangsu Agric Sci, 2020, 48(16): 67-73.
3	章秀福, 朱德峰. 中国直播稻生产现状与前景展望 [J]. 中国稻米, 1996, 2(5): 1-4.
	Zhang XF, Zhu DF. Present situation and prospect of direct seeding rice production in China [J]. China Rice, 1996, 2(5): 1-4.
4	邱龙, 马崇烈, 刘博林, 等. 耐除草剂转基因作物研究现状及发展前景 [J]. 中国农业科学, 2012, 45(12): 2357-2363.
	Qiu L, Ma CL, Liu BL, et al. Current situation of research on transgenic crops with herbicide tolerance and development prospect [J]. Sci Agric Sin, 2012, 45(12): 2357-2363.
5	Zong Y, Liu YJ, Xue CX, et al. An engineered prime editor with enhanced editing efficiency in plants [J]. Nat Biotechnol, 2022, 40(9): 1394-1402.
6	Fartyal D, Agarwal A, James D, et al. Co-expression of P173S mutant rice EPSPS and igrA genes results in higher glyphosate tolerance in transgenic rice [J]. Front Plant Sci, 2018, 9: 144.
7	Komor AC, Kim YB, Packer MS, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage [J]. Nature, 2016, 533(7603): 420-424.
8	Gaudelli NM, Komor AC, Rees HA, et al. Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage [J]. Nature, 2017, 551(7681): 464-471.
9	Tong HW, Wang XC, Liu YH, et al. Programmable A-to-Y base editing by fusing an adenine base editor with an N-methylpurine DNA glycosylase [J]. Nat Biotechnol, 2023, 41(8): 1080-1084.
10	Komor AC, Zhao KT, Packer MS, et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C: G-to-T: a base editors with higher efficiency and product purity [J]. Sci Adv, 2017, 3(8): eaao4774.
11	Li J, Xu RF, Qin RY, et al. Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants [J]. Mol Plant, 2021, 14(2): 352-360.
12	辛洁, 徐小博, 王磊, 等. ALS抑制剂类除草剂的抗性研究概述 [J]. 安徽农业科学, 2019, 47(4): 18-21.
	Xin J, Xu XB, Wang L, et al. Study on the resistance of ALS inhibitor herbicides [J]. J Anhui Agric Sci, 2019, 47(4): 18-21.
13	Wang MG, Lu YM, Botella JR, et al. Gene targeting by homology-directed repair in rice using a geminivirus-based CRISPR/Cas9 system [J]. Mol Plant, 2017, 10(7): 1007-1010.
14	Sun YW, Zhang X, Wu CY, et al. Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase [J]. Mol Plant, 2016, 9(4): 628-631.
15	Shimatani Z, Kashojiya S, Takayama M, et al. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion [J]. Nat Biotechnol, 2017, 35(5): 441-443.
16	Kuang YJ, Li SF, Ren B, et al. Base-editing-mediated artificial evolution of OsALS1 in planta to develop novel herbicide-tolerant rice germplasms [J]. Mol Plant, 2020, 13(4): 565-572.
17	Fan TT, Cheng YH, Wu YC, et al. High performance TadA-8e derived cytosine and dual base editors with undetectable off-target effects in plants [J]. Nat Commun, 2024, 15(1): 5103.
18	Chen YY, Wang ZP, Ni HW, et al. CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis [J]. Sci China Life Sci, 2017, 60(5): 520-523.
19	Li YM, Zhu JJ, Wu H, et al. Precise base editing of non-allelic acetolactate synthase genes confers sulfonylurea herbicide resistance in maize [J]. Crop J, 2020, 8(3): 449-456.
20	Tian SW, Jiang LJ, Cui XX, et al. Engineering herbicide-resistant watermelon variety through CRISPR/Cas9-mediated base-editing [J]. Plant Cell Rep, 2018, 37(9): 1353-1356.
21	Veillet F, Perrot L, Chauvin L, et al. Transgene-free genome editing in tomato and potato plants using Agrobacterium-mediated delivery of a CRISPR/Cas9 cytidine base editor [J]. Int J Mol Sci, 2019, 20(2): 402.
22	Wu J, Chen C, Xian GY, et al. Engineering herbicide-resistant oilseed rape by CRISPR/Cas9-mediated cytosine base-editing [J]. Plant Biotechnol J, 2020, 18(9): 1857-1859.
23	Zhang R, Liu J, Chai Z, et al. Generation of herbicide tolerance traits and a new selectable marker in wheat using base editing [J]. Nat Plants, 2019, 5(5): 480-485.
24	陈晓军, 吴凯伸, 惠建, 等. 基于基因编辑技术创制高亚油酸水稻材料 [J]. 中国农业科学, 2021, 54(14): 2931-2940.
	Chen XJ, Wu KS, Hui J, et al. Production of high linoleic acid rice by genome editing [J]. Sci Agric Sin, 2021, 54(14): 2931-2940.
25	陈晓军, 王敬东, 宋海丽, 等. 一种简单、极快的植物叶片DNA提取方法 [J]. 种子, 2018, 37(11): 26-29, 34.
	Chen XJ, Wang JD, Song HL, et al. A simple and rapid method of DNA extraction from plant leaf [J]. Seed, 2018, 37(11): 26-29, 34.
26	余铮, 邓莉立, 谭显胜, 等. 20%双草醚可湿性粉剂对水稻直播田杂草的防除效果及安全性评价 [J]. 杂草学报, 2017, 35(3): 38-42.
	Yu Z, Deng LL, Tan XS, et al. Efficacy and selectivity of bispyribac-sodium 20% WP in direct-seeded rice fields [J]. J Weed Sci, 2017, 35(3): 38-42.
27	Croughan TP. Resistance to acetohydroxyacid synthase-inhibiting herbicides in rice: US20090025108 [P]. 2009-01-22.
28	Shoba D, Raveendran M, Manonmani S, et al. Development and genetic characterization of A novel herbicide (imazethapyr) tolerant mutant in rice (Oryza sativa L.) [J]. Rice, 2017, 10(1): 10.
29	Chen L, Gu G, Wang CX, et al. Trp548Met mutation of acetolactate synthase in rice confers resistance to a broad spectrum of ALS-inhibiting herbicides [J]. Crop J, 2021, 9(4): 750-758.
30	Okuzaki A, Shimizu T, Kaku K, et al. A novel mutated acetolactate synthase gene conferring specific resistance to pyrimidinyl carboxy herbicides in rice [J]. Plant Mol Biol, 2007, 64(1-2): 219-224.
31	Kawai K, Kaku K, Izawa N, et al. A novel mutant acetolactate synthase gene from rice cells, which confers resistance to ALS-inhibiting herbicides [J]. J Pestic Sci, 2007, 32(2): 89-98.
32	Zafar K, Khan MZ, Amin I, et al. Employing template-directed CRISPR-based editing of the OsALS gene to create herbicide tolerance in Basmati rice [J]. AoB Plants, 2023, 15(2): plac059.
33	Ren QR, Sretenovic S, Liu SS, et al. PAM-less plant genome editing using a CRISPR-SpRY toolbox [J]. Nat Plants, 2021, 7(1): 25-33.
34	Wang FQ, Xu Y, Li WQ, et al. Creating a novel herbicide-tolerance OsALS allele using CRISPR/Cas9-mediated gene editing [J]. Crop J, 2021, 9(2): 305-312.
35	Nishizawa-Yokoi A, Mikami M, Toki S. A universal system of CRISPR/Cas9-mediated gene targeting using all-in-one vector in plants [J]. Front Genome Ed, 2020, 2: 604289.
36	Zhang R, Chen S, Meng XB, et al. Generating broad-spectrum tolerance to ALS-inhibiting herbicides in rice by base editing [J]. Sci China Life Sci, 2021, 64(10): 1624-1633.
37	Walton RT, Christie KA, Whittaker MN, et al. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants [J]. Science, 2020, 368(6488): 290-296.

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