水稻OsAlR1基因耐铝性功能研究

doi:10.16819/j.1001-7216.2025.240510

摘要/Abstract

摘要：

【目的】随着土壤的酸化加剧，酸性土壤中的铝毒严重限制了水稻的生长发育，挖掘耐铝毒基因资源并解析其分子功能，培育耐铝毒品种，可保障酸性土壤上水稻的产量。【方法】利用CRISPR/Cas9技术构建敲除载体，采用同源重组和Golden Gate无缝克隆方法构建过表达载体，遗传转化获得转基因材料。筛选纯合过表达株系和敲除株系进行耐铝性表型分析并测定活性氧(ROS)含量及抗氧化酶活性。【结果】qRT-PCR结果表明，OsAlR1受铝胁迫诱导表达。敲除OsAlR1影响水稻根系发育。在铝处理后，相较于野生型，osalr1-3、osalr1-6株系积累了更多的Al³⁺及ROS，OE-OsAlR1-5、OE-OsAlR1-8株系积累的Al³⁺及ROS较少。OsAlR1的敲除会导致根中谷胱甘肽(GSH)的积累和SOD等抗氧化酶活性降低。qRT-PCR结果表明，在铝处理后调控苯丙烷生物合成途径的OsPAL7、OsHCT4、OsCCR18、Os02g0467600、PRX82和Os06g0522300在敲除突变体中表达量变化显著。【结论】OsAlR1受到铝胁迫的诱导，OsAlR1通过提高GSH的含量和SOD的活性来降低ROS对水稻的氧化胁迫。OsAlR1可能通过调控苯丙烷生物合成途径来影响水稻耐铝性。

关键词: 水稻, 铝胁迫, 根系, ROS, 抗氧化酶

Abstract:

【Objective】 With the aggravation of soil acidification, aluminum (Al) toxicity in acidic soil seriously limits the growth and development of rice. Mining Al-tolerant gene resources, analyzing their molecular functions, and breeding Al-tolerant varieties can ensure rice yield in acidic soil. 【Method】 CRISPR/Cas9 technology was used to construct knockout vectors. The overexpression vector was constructed by homologous recombination and Golden Gate seamless cloning. Transgenic materials were obtained by genetic transformation. Homozygous overexpression lines and knockout lines were screened for Al tolerance phenotype analysis and determination of reactive oxygen species (ROS) content and antioxidant enzyme activities.【Result】 qRT-PCR results showed that OsAlR1 expression was induced by Al stress. Knockout of OsAlR1 affected root development in rice. After Al treatment, compared with the wild type, the osalr1-3 and osalr1-6 lines accumulated more Al³⁺ and ROS, while the OE-OsAlR1-5 and OE-OsAlR1-8 lines accumulated less Al³⁺ and ROS. Knockout of OsAlR1 led to decreased glutathione (GSH) content and superoxide dismutase (SOD) activity in roots. qRT-PCR results revealed that genes regulating the phenylpropanoid biosynthesis pathway (OsPAL7, OsHCT4, OsCCR18, Os02g0467600, PRX82, and Os06g0522300) showed significant expression changes in the knockout lines after Al treatment.【Conclusion】 OsAlR1 expression is induced by Al stress, and OsAlR1 alleviates ROS-mediated oxidative stress by increasing GSH content and SOD activity. OsAlR1 may affect Al tolerance in rice by regulating the phenylpropanoid biosynthesis pathway.

Key words: rice, aluminum stress, roots, ROS, antioxidant enzyme

王镜博, 苏畅, 冯晶, 姜思旭, 徐海, 崔志波, 赵明辉. 水稻OsAlR1基因耐铝性功能研究[J]. 中国水稻科学, 2025, 39(5): 615-623.

WANG Jingbo, SU Chang, FENG Jing, JIANG Sixu, XU Hai, CUI Zhibo, ZHAO Minghui. Functional Study on Aluminum Tolerance of OsAlR1 Gene in Rice[J]. Chinese Journal OF Rice Science, 2025, 39(5): 615-623.

图/表 8

表1 本研究所用引物

Table 1. Primer sequences used in this study

引物名称 Primer name	序列 Primer sequence (5'-3')	用途 Usage
OsAlR1-F	AACACGGGGGACTTTGCAACATGGGCAACTCGCTGGCGTTG	基因扩增 Gene amplification
OsAlR1-R	TCCTCGCCCTTCACGATACACGCCTCATAGATGGTATCAAGGGC	基因扩增 Gene amplification
OsAlR1-gRNA-F	GGCAGCGTTGCGGTGCGTCTGTGG	CRISPR/Cas9载体构建 CRISPR/Cas9 vector construction
OsAlR1-gRNA-R	AAACCCACAGACGCACCGCAACGC	CRISPR/Cas9载体构建 CRISPR/Cas9 vector construction
OsAlR1-seq-F	GGAGAGAACACGGGGGAC	鉴定 Identification
OsAlR1-seq-R	GTGCAGATGAACTTGAGG	鉴定 Identification
OsAlR1-OE-F	GGAGAGAACACGGGGGAC	表达量分析 Expression analysis
OsAlR1-OE-R	GTGCAGATGAACTTGAGG
OsAlR1-KO-F	ATGGGCAACTCGCTGGCGTTG
OsAlR1-KO-R	CGCCTCATAGATGGTATCAAGGGC
OsAlR1-qPCR-F	AGCTGATGATCGAGGCGC
OsAlR1-qPCR-R	CTTGCTCCGCCCTTTCTTG
OsPAL7-qPCR-F	CATCGGGAAGCTCATGTTCG
OsPAL7-qPCR-R	CTTGAAGCCGTAGTCCAAGC
OsHCT4-qPCR-F	GGTCCGGCGATCATGTACTA
OsHCT4-qPCR-R	GCGAACACCTTCCTGAACTC
Os02g0467600-qPCR-F	CGCAGAGCTTCGAGTACAAC
Os02g0467600-qPCR-R	CACGGCACTTGTTGAGGTAG
OsCCR18-qPCR-F	CTGATGACGCTGCCAAGAAC
OsCCR18-qPCR-R	ATCATCTTCTCCGGGTCGTC
PRX82-qPCR-F	CGAGGAGTTCACGGAGAGTT
PRX82-qPCR-R	AGCAGTCGTGGAAGAAGAGG
Os06g0522300-qPCR-F	CTGACCAGGAGCTCTACACC
Os06g0522300-qPCR-R	CTCTTGTGAAGTCGGCGAAG

图1 铝胁迫对OsAlR1表达量的影响

Fig. 1. Effects of Al treatment on the expression of OsAlR1

图2 转基因植株的阳性鉴定 A: 过表达株系阳性鉴定; B: 敲除株系阳性鉴定; C: 过表达株系表达量分析; D: 敲除株系靶点解码分析。M: Trans 1K; P: 阳性对照。

Fig. 2. Identification of transgenic plants A, Identification of overexpressing lines; B, Identification of knockout lines; C, Expression analysis of the overexpression lines; D, Target site analysis of knockout lines. M, Trans 1K; P, Positive control.

图3 转基因植株的根长测定 (比例尺=10 cm) A：敲除株系对照组根长；B：敲除株系实验组根长；C：敲除株系在铝处理前后根长比较；D：过表达株系对照组根长；E：过表达株系实验组根长; F：过表达株系在Al处理前后根长比较；CK：铝处理前; Al：铝处理后。*表示P值小于0.05。下同。

Fig. 3. Root length identification of transgenic plants (Scale Bar=10 cm) A, Root length of control groups of knockout lines; B, Root length of experimental groups of knockout lines; C, Comparison of root length of knockout lines before and after Al treatment; D, Root length of control groups of overexpression lines; E, Root length of experimental groups of overexpression lines; F, Comparison of root length of overexpression lines before and after Al treatment; CK, Before Al treatment; Al, After Al treatment. * indicates significant difference at the 0.05 probability level. The same below.

图4 转基因植株Al3+含量测定

Fig. 4. Al3+ content determination of transgenic plants

图5 转基因植株根系ROS的积累

Fig. 5. ROS production in transgenic plants roots

图6 转基因植株根系抗氧化酶活性测定

Fig. 6. ROS production in transgenic plants roots

图7 OsAlR1转基因水稻耐铝相关基因的RT-qPCR分析

Fig. 7. RT-qPCR analysis of Al tolerance genes in OsAlR1 transgenic lines

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