敲除OsNramp5基因创制低镉优质粳稻新材料的应用评价

doi:10.16819/j.1001-7216.2023.220503

中国水稻科学 ›› 2023, Vol. 37 ›› Issue (1): 16-28.DOI: 10.16819/j.1001-7216.2023.220503

敲除OsNramp5基因创制低镉优质粳稻新材料的应用评价

裴峰¹, 王广达¹, 高鹏¹, 冯志明¹^,², 胡珂鸣¹^,², 陈宗祥¹^,², 陈红旗³, 崔傲⁴, 左示敏¹^,²^,⁵()

¹扬州大学江苏省作物基因组学和分子育种重点实验室/植物功能基因组学教育部重点实验室，江苏扬州 225009
²扬州大学江苏省粮食作物现代产业技术协同创新中心/江苏省作物遗传生理重点实验室，江苏扬州 225009
³中国水稻研究所水稻生物学国家重点实验室，杭州 311400
⁴镇江禾下土农业科技有限公司，江苏镇江 212008
⁵扬州大学教育部农业与农产品安全国际合作联合实验室，江苏扬州 225009

收稿日期:2022-05-06 修回日期:2022-07-31 出版日期:2023-01-10 发布日期:2023-01-10
通讯作者: 左示敏
基金资助:
江苏省重点研发计划资助项目(BE2019339);江苏省农业重大新品种创制项目(PZCZ201703);扬州市重点研发计划资助项目(现代农业YZ2020031(现代农业YZ2020031)

Evaluation of New japonica Rice Lines with Low Cadmium Accumulation and Good Quality Generated by Knocking Out OsNramp5

PEI Feng¹, WANG Guangda¹, GAO Peng¹, FENG Zhiming¹^,², HU Keming¹^,², CHEN Zongxiang¹^,², CHEN Hongqi³, CUI Ao⁴, ZUO Shimin¹^,²^,⁵()

¹Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding / Key Laboratory of Plant Functional Genomics of Ministry of Education, College of Agriculture, Yangzhou University, Yangzhou 225009, China
²Jiangsu Collaborative Innovation Center for Modern Industrial Technology of Grain Crops / Jiangsu Key Laboratory of Crop Genetics and Physiology, Yangzhou University, Yangzhou 225009, China
³State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 311400, China
⁴Zhenjiang Hexiatu Agricultural Science and Technology Co., Ltd, Zhenjiang 212008, China
⁵Joint International Research Laboratory of Agriculture and Agri-Product Safety, Ministry of Education of China, Yangzhou University, Yangzhou 225009, China

Received:2022-05-06 Revised:2022-07-31 Online:2023-01-10 Published:2023-01-10
Contact: ZUO Shimin

摘要/Abstract

摘要： 目的探究在粳稻中敲除OsNramp5基因对镉等金属元素积累、产量和品质的影响，为科学高效地生产优质健康粳米提供新材料和理论参考。方法利用CRISPR/Cas9技术，在优良食味高产粳稻品种南粳46背景下对OsNramp5进行基因敲除，结合标记辅助选择，获得了无外源基因的OsNramp5敲除突变系，考查其产量和品质性状变化特征，以及在不同镉含量土壤中和锌肥、硒肥喷施后的籽粒镉、锰、硒等七种金属元素的含量变化。结果在T₀代获得了5种OsNramp5不同变异类型的转基因植株，设计开发了其中3种突变型的特异分子标记，进而结合标记辅助选择，在各植株的T₂代中获得不含潮霉素选择标记基因和CAS9蛋白基因的纯合敲除系。与野生型相比，无论在高浓度还是低浓度镉含量土壤中，敲除系整株包括籽粒中的镉含量和地上部组织中锰含量均显著低于南粳46；敲除系的株高略微下降，穗粒数显著降低并导致其单株产量明显下降，但在低分蘖肥处理中产量下降未达显著。敲除系稻米外观总体好于对照，蛋白质和直链淀粉含量明显高于对照，导致其食味值明显降低。敲除系籽粒中铜、锰和硒的含量显著低于对照，但在施用锌肥和硒肥后可以显著提高锌和硒的含量，同时有利于进一步降低籽粒镉的含量。施硒有利于改善稻米外观但施锌则相反，喷施两种肥料都会降低稻米食味值，不利于食味品质提升。敲除系在极端高/低温条件下的结实率变化与对照无异，但对纹枯病的抗性显著降低。结论在优质高产粳稻品种中获得了无标记OsNramp5基因敲除系，证明其在生产低镉富硒富锌安全营养健康型稻米中具有重要价值，但在产量和食味品质上则有不同程度的下降。

关键词: 粳稻, 低镉, 基因编辑, 品质, 产量, 育种价值

Abstract:

【Objective】 It is crucial to elucidate the effects of OsNramp5 gene knockout on cadmium and other metal elements accumulation, yield and quality in japonica rice, which can provide new materials and theoretical references for scientific and efficient production of high-quality and green japonica rice.【Methods】 Using CRISPR/Cas9 technology, the OsNramp5 was knocked out under the background of Nanjing 46 (NJ46), a japonica rice variety with good taste and high yield. Combined with molecular marker-assisted selection, some OsNramp5 knockout lines without exogenous genes were obtained. The changes in yield and quality traits and seven metal elements contents such as cadmium, manganese and selenium in grains after spraying zinc fertilizer or selenium fertilizer and cultivated in soils with different cadmium content were investigated. 【Results】 Five transgenic plants with different mutations of OsNramp5 were obtained in the T₀ generation, and specific molecular markers of three of them were designed. Then, combined with marker-assisted selection, homozygous knockout lines without hygromycin selective marker gene and CAS9 protein gene were obtained in the T₂ generation of each plant. Compared with the wild type, the cadmium content in the whole plant including the grain of the knockout lines and the manganese content in shoots were significantly lower than those of the wild type in both high and low concentrations of cadmium soil. The plant height and grain number per panicle of the knockout lines decreased slightly, and the yield per plant decreased significantly in the knockout lines at high and medium tillering fertilizer levels while no changes were observed at the low tillering fertilizer level. The appearance of the knockout lines was better than that of the control, and the contents of protein and amylose were significantly higher than those of the control. The contents of copper, manganese and selenium in grains of knockout lines were significantly lower than those of the control NJ46, but zinc and selenium fertilizer could significantly increase the contents of zinc and selenium, and further reduce the content of cadmium in grains. Application of selenium was beneficial to rice appearance quality, but application of zinc was contrary. Spraying both fertilizers reduced the edible value of rice, worsening taste quality. The change of seed setting rate of the knockout lines under extreme high and low temperature conditions was the same as that of the control, but the resistance level to rice sheath blight was significantly reduced.【Conclusion】 We obtained marker-free OsNramp5 knockout lines under the background of japonica variety with high quality and yield, and proved that OsNramp5 was of great value in the production of low-cadmium, Se- and zinc-rich, though their yield and eating quality were decreased to different extents.

Key words: japonica rice, low cadmium accumulation, gene editing, grain quality, grain yield, breeding value

裴峰, 王广达, 高鹏, 冯志明, 胡珂鸣, 陈宗祥, 陈红旗, 崔傲, 左示敏. 敲除OsNramp5基因创制低镉优质粳稻新材料的应用评价[J]. 中国水稻科学, 2023, 37(1): 16-28.

PEI Feng, WANG Guangda, GAO Peng, FENG Zhiming, HU Keming, CHEN Zongxiang, CHEN Hongqi, CUI Ao, ZUO Shimin. Evaluation of New japonica Rice Lines with Low Cadmium Accumulation and Good Quality Generated by Knocking Out OsNramp5[J]. Chinese Journal OF Rice Science, 2023, 37(1): 16-28.

图/表 9

图1 极端温度条件的温度变化曲线 A―极端高温的日温度变化曲线；B―极端低温的日温度变化曲线。

Fig. 1. Temperature curves for extreme temperature conditions. A, Diurnal temperature curve of extreme high temperature; B, Diurnal temperature curve of extreme low temperature.

图2 OsNramp5敲除系的构建和基因型检测 A―靶点序列在OsNramp5上的位置。B―pC1300-Cas9-OsNramp5-sgRNA表达载体的结构。C―T0代植株与野生型的基因序列比对分析。黑色阴影为相同序列，灰色阴影为靶点序列，“+”表示插入，“-”表示缺失，“WT”表示野生型。D―T0代植株与野生型的氨基酸比对分析。序列左侧和右侧数字表示该氨基酸序列在蛋白中的位置；黑色阴影为与野生型相同序列，“-”表示缺失，“WT”表示野生型。E―各突变型的特异分子标记开发及PCR检测。图上方分别标注有检测的家系号和使用的特异PCR标记名称；#1和#2号系均是开发的CAPS标记，泳道1为经过测序确认的纯合突变型，泳道2为野生型，泳道3-8分别表示T2世代的不同单株，其中双带表示杂合基因型；#5号系开发的是InDel标记，电泳图中泳道1-6为纯合突变型，泳道7为野生型；使用的分子量标记为DL 2000（下同）。F―部分纯合突变型株系的转基因成分检测。泳道1和2分别为阳性和阴性对照，泳道3~11为不同株系的检测结果，其中泳道7、10和11分别对应nramp5ko-1、-2和-3。

Fig. 2. Construction and genotyping of OsNramp5 knockout lines. Note: (A) Position of the target sequence in OsNramp5; (B) Construction of the vector of pC1300-Cas9-OsNramp5-sgRNA;(C) Comparative analysis of gene sequences between T0 generation plants and wild type. Black shadows represent the same sequences, gray shadows represent the target sequence, ‘+’ Insertion, ‘-’ Deletion, ‘WT’ Wild type;(D) Comparative analysis of amino acids between T0 generation plants and wild type. The numbers to the left and right of the sequence indicate the position of the amino acid sequence in the protein. The black shadows represent the same sequences as the wild type, ‘-’ Deletion, ‘WT’ Wild type ;(E) Development of molecular markers specific to three different variants lines. Different mutant lines and their specific markers were marked at the top of the figure. CAPS markers were developed for mutant lines #1 and #2, and in their electrophoretic picture, the lane 1 represents homozygous mutant that was confirmed by sequencing. The lane 2 indicates the wild type and remaining lanes 3~8 represent different individual plants from T2 population. InDel marker was developed for genotyping mutant lines #5, and the lane 7 and the remaining lanes indicate wild type and different individual T2 plants, respectively. DL 2000 was used as the molecular marker (Same as below). (F) Detection of transgenic components for some homozygous mutant plant lines. Lanes 1 and 2 are positive and negative controls, respectively. Lanes 3~11 represent different homozygous mutant plant lines, and among them, lanes 7, 10 and 11 correspond to nramp5ko-1, -2 and -3 respectively.

图3 OsNramp5敲除系植株的株型及镉和锰的含量分析 A―敲除系与野生型的株型。比例尺=50 cm；B―敲除系与野生型的株高；C―敲除系与野生型在不同镉浓度下不同部位的镉含量。C0为对照，C1为1.2 mg/kg外源镉，C2为2.0 mg/kg外源镉；D―敲除系与野生型在不同处理下地上部的锰含量。C0为对照，C0+Mn为喷施锰肥处理。数据为平均数±标准差（n=3）。*代表与WT有显著性差异（P<0.05）（t检验）。

Fig. 3. Whole plant and analysis of cadmium and manganese contents in OsNramp5 knockout lines. A,Whole plant of the wild type and homozygous mutant lines. Scale = 50 cm. B, Plant height of knockout lines and wild type. C, Cadmium content in different parts of knockout lines and wild type under different cadmium concentrations. ‘C0’,control; ‘C1’, 1.2 mg/kg cadmium; ‘C2’, 2.0 mg/kg cadmium; D, Manganese content in shoots of knockout lines and wild type under different treatments. ‘C0’, control; ‘C0+Mn’, spraying manganese fertilizer treatment. Data are shown as means ± SD(n=3). * represent a significant difference from WT (P<0.05) (t-test).

表1 OsNramp5敲除系与对照在不同分蘖肥处理间的理论产量及其构成因素比较

Table 1. Comparison of theoretical yield and its components between OsNramp5 knockout lines and control at different tillering fertilizer levels.

分蘖肥处理 Tillering fertilizer level	株系 Line	有效穗 Effective panicle	每穗粒数 Grains per panicle	结实率 Seed setting rate/ %	千粒重 1000-grain weight / g	理论产量Theoretical yield/(kg·hm⁻²)
低肥 Low (37.5 kg/hm²)	nramp5ko-1	9.6±1.1 b	125.4±3.9 e	96.12±0.96 a	24.36±0.15 bc	8541.75
	nramp5ko-2	9.9±2.1 ab	123.2±3.3 e	96.59±0.98 a	24.47±0.47 abc	8735.70
	WT	7.8±1.1 c	156.0±4.9 b	95.71±0.95 a	24.82±0.05 a	8759.25
正常 Normal (112.5 kg/hm²)	nramp5ko-1	9.9±1.1 ab	138.4±1.9 cd	91.27±1.43 c	24.42±0.03 abc	9254.10
	nramp5ko-2	9.6±1.9 b	141.4±2.1 cd	92.69±1.73 bc	24.41±0.07 abc	9306.90
	WT	9.9±1.6 ab	163.8±4.2 a	95.44±0.77 a	24.73±0.04 ab	11 598.15
高肥 High (187.5 kg/hm²)	nramp5ko-1	11.2±1.0 a	137.0±1.7 d	92.15±2.60 bc	23.02±0.08 d	9863.40
	nramp5ko-2	11.1±1.8 ab	141.8±2.3 c	91.33±3.39 c	23.19±0.03 d	10 101.75
	WT	10.8±1.6 ab	165.2±3.2 a	93.52±1.23 b	24.23±0.09 c	12 251.25

图4 三种施肥处理下OsNramp5敲除系和野生型籽粒中的元素含量 A、B和C分别是正常施肥、增施锌肥和增施硒肥条件下的籽粒中铜、铁、锌、锰、镉和硒元素的含量。数据为平均数±标准差（n=3）。 *代表连线之间具有显著性差异（P<0.05）（t检验）。

Fig. 4. Element contents in grains of OsNramp5 knockout lines and wild type under three fertilization treatments. A, B and C were contents of copper, iron, zinc, manganese, cadmium and selenium in grains under normal fertilization, zinc fertilization and selenium fertilization respectively. Data are shown as means ± SD(n=3). * represents significant differences between lines(P<0.05) (t-test).

表2 OsNramp5-ko敲除系在不同施肥处理中的稻米加工和品质相关指标差异比较

Table 2. Comparison of rice processing and quality related indexes of OsNramp5-ko knockout lines under different fertilization treatments.

施肥方式Fertilization treatment	株系 Line	糙米率 Brown rice rate /%	精米率 Milled rice rate /%	整精米率 Head milled rice rate /%	垩白粒率 Chalky rice rate/%	垩白度 Chalkiness /%
正常 Conventional	nramp5ko-1	84.89±0.17 b	72.20±0.50 b	67.29±0.70 b	6.6±0.2 f	2.0±0.1 d
	nramp5ko-2	85.12±0.27 ab	73.44±1.97 ab	67.31±1.59 b	7.7±0.2 e	1.9±0.1 d
	WT	84.91±0.23 ab	73.86±0.83 a	69.49±0.74 ab	8.7±0.1 c	2.3±0.1 c
锌肥 +Zn	nramp5ko-1	84.91±0.29 ab	71.95±0.39 b	67.63±1.58 b	9.4±0.3 b	2.7±0.2 b
	nramp5ko-2	84.95±0.08 ab	72.92±0.86 ab	67.61±1.15 b	8.2±0.1 d	2.5±0.1 bc
	WT	84.82±0.26 b	73.38±1.00 ab	69.85±1.99 a	11.8±0.2 a	3.4±0.2 a
硒肥 +Se	nramp5ko-1	85.24±0.15 a	73.10±1.69 ab	67.72±1.17 b	4.3±0.1 h	1.1±0.1 e
	nramp5ko-2	85.22±0.18 a	72.53±0.26 ab	67.66±2.85 b	4.6±0.1 h	1.2±0.1 e
	WT	84.94±0.27 ab	73.30±0.68 ab	70.08±0.42 a	5.5±0.2 g	1.4±0.1 e

图5 不同肥料处理下OsNramp5-KO及对照的精米外观

Fig. 5. Milled rice appearance of OsNramp5-KO lines and control under different fertilizer treatments.

表3 OsNramp5-ko敲除系在不同施肥处理中的稻米营养品质和食味品质相关指标差异比较

Table 3. Comparison of OsNramp5-ko knockout lines in rice nutritional quality and food quality indexes under different fertilization treatments.

施肥方式 Fertilization treatment	株系 Line	蛋白质含量 Protein content / %	直链淀粉含量 Amylose content/%	胶稠度 Gel consistency /mm	硬度Hardness	黏度viscosity	平衡度Balance	食味值 Taste value
正常 Conventional	nramp5ko-1	7.55±0.02 a	9.35±0.00 d	75.5±0.5 a	6.1±0.1 a	7.3±0.1 d	7.3±0.1 e	74.6±0.9 de
	nramp5ko-2	7.24±0.01 b	9.87±0.00 c	75.5±0.5 a	5.8±0.0 c	7.9±0.3 abc	7.9±0.2 bcd	77.9±1.4 bcd
	WT	6.82±0.01 c	10.20±0.04 b	77.5±0.5 a	5.6±0.0 d	8.4±0.1 a	8.4±0.1 a	81.4±0.8 a
锌肥 +Zn	nramp5ko-1	7.30±0.00 b	10.38±0.00 ab	77.5±0.5 a	5.9±0.1 bc	8.2±0.2 ab	8.2±0.3 ab	79.8±1.9 ab
	nramp5ko-2	7.47±0.02 a	10.60±0.08 a	77.0±1.0 a	5.9±0.1 bc	8.1±0.2 ab	8.0±0.1 abc	78.5±1.0 abc
	WT	6.89±0.04 c	10.72±0.18 a	78.5±0.5 a	5.8±0.1 c	7.7±0.3 bcd	7.8±0.2 bcde	77.3±1.7 bcde
硒肥 +Se	nramp5ko-1	7.22±0.04 b	10.42±0.11 ab	69.0±1.0 b	6.0±0.2 ab	7.4±0.5 cd	7.4±0.5 de	75.2±3.0 de
	nramp5ko-2	7.27±0.04 b	10.53±0.15 ab	69.5±1.5 b	6.1±0.0 a	7.4±0.2 cd	7.3±0.1 e	74.4±0.7 e
	WT	7.25±0.02 b	10.42±0.04 ab	69.0±1.0 b	5.9±0.0 bc	7.5±0.1 cd	7.5±0.0 cde	75.8±0.2 cde

图6 OsNramp5敲除系在极端温度及纹枯病菌接种处理下与野生型的抗逆性差异 A―敲除系和野生型在极端高温和低温胁迫下的结实率；B―敲除系和野生型的离体接种纹枯病的发病情况。数据为平均数±标准差（n=3）。*代表与WT有显著性差异（P<0.05）（t检验）。

Fig 6. Stress resistance difference between OsNramp5 knockout lines and wild-type under extreme high and low temperature and rice sheath blight inoculation. A, Seed setting rate of knockout lines and wild type under extreme high and low temperature stress; B, Incidence of rice sheath blight of knockout lines and wild type after inoculation in vitro. Data are shown as means ± SD(n=3). * represents a significant difference from WT(P<0.05) (t-test).

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敲除OsNramp5基因创制低镉优质粳稻新材料的应用评价

Evaluation of New japonica Rice Lines with Low Cadmium Accumulation and Good Quality Generated by Knocking Out OsNramp5

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