基于CRISPR/Cas9技术的水稻<i>OsASL1.1</i>基因突变体创制及干旱胁迫下的表型分析

doi:10.7668/hbnxb.20195973

摘要/Abstract

摘要：

为研究OsASL1.1基因的表达特性及在干旱胁迫响应中的作用,分析OsASL1.1蛋白的亚细胞定位和OsASL1.1基因启动子区顺式作用元件,并利用实时荧光定量PCR(qRT-PCR)分析OsASL1.1基因在脱落酸(ABA)、甘露醇、聚乙二醇6000(PEG6000)下的表达模式,同时以Kitaake为背景采用CRISPR/Cas9基因编辑技术构建Cas9-OsASL1.1突变体,分析突变体的突变类型,并对突变体进行ABA敏感性和抗旱性分析。结果表明,OsASL1.1蛋白主要在质体中表达。OsASL1.1基因启动子区含有低温响应、植物激素(ABA、生长素)响应等顺式作用元件,其中,ABA响应顺式作用元件(ABRE)有5个。OsASL1.1基因受ABA、甘露醇、PEG6000诱导表达。获得了Cas9-6和Cas9-12两个纯合的突变类型,Cas9-6有17 bp的插入,导致翻译提前终止;Cas9-12有12 bp的缺失,导致缺失4个氨基酸。ABA处理下,Cas9-OsASL1.1突变体和野生型的主根伸长都受到了抑制,但Cas9-OsASL1.1突变体受到的抑制程度低于野生型。150 mmol/L甘露醇处理下,Cas9-6和Cas9-12突变体的存活率均显著低于野生型,以Cas9-6的存活率最低。综上,Cas9-OsASL1.1突变体对ABA的敏感性降低,对干旱的抗性降低,即OsASL1.1 基因功能丧失降低了水稻对ABA的敏感性和抗旱性。

关键词: 水稻, OsASL1.1, CRISPR/Cas9, 突变体, 干旱胁迫

Abstract:

To investigate the expression characteristics of OsASL1.1 and its role in the drought stress response,the subcellular localization of OsASL1.1 protein and cis-acting elements in the promoter region of OsASL1.1 gene were analyzed.Additionally,Real-time Quantitative PCR(qRT-PCR)was used to examine the expression patterns of OsASL1.1 in response to abscisic acid(ABA),mannitol,and polyethylene glycol 6000(PEG6000).CRISPR/Cas9 gene editing technology was employed to generate Cas9-OsASL1.1 mutants with Kitaake as background,and the mutation types were characterized.The abscisic acid(ABA)sensitivity and drought tolerance of mutants were analyzed.The results indicated that OsASL1.1 protein was primarily localized in plastids.The promoter region of OsASL1.1 contained cis-acting elements responsive to low temperatures and plant hormones(ABA and auxin),including five ABA responsive cis-acting elements(ABREs).OsASL1.1 expression was induced by ABA,mannitol and PEG6000.Two homozygous mutant types,Cas9-6 and Cas9-12,were generated.Cas9-6 harbored a 17 bp insertion that caused premature translation termination,whereas Cas9-12 had a 12 bp deletion that resulted in the loss of four amino acids.Under ABA treatment,root elongation of Cas9-OsASL1.1 mutants and wild type were inhibited;however,the inhibition was less severe in the mutants than that in the wild type.Under 150 mmol/L mannitol treatment,the survival rates of both Cas9-6 and Cas9-12 mutants were significantly lower than that of wild type,with Cas9-6 showing the lowest survival rate.In summary,the Cas9-OsASL1.1 mutants show reduced sensitivity to ABA and decreased drought tolerance,indicating that the loss of OsASL1.1 function diminishes rice sensitivity to ABA and drought tolerance.

Key words: Rice, OsASL1.1, CRISPR/Cas9, Mutant, Drought stress

中图分类号:

胡鑫, 陈聪, 端木凡青, 李颖, 董全师, 杜长青. 基于CRISPR/Cas9技术的水稻OsASL1.1基因突变体创制及干旱胁迫下的表型分析[J]. 华北农学报, 2026, 41(1): 22-29. doi: 10.7668/hbnxb.20195973.

HU Xin, CHEN Cong, DUANMU Fanqing, LI Ying, DONG Quanshi, DU Changqing. Creation of Rice OsASL1.1 Mutants Based on CRISPR/Cas9 Technology and Phenotypic Analysis under Drought Stress[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(1): 22-29. doi: 10.7668/hbnxb.20195973.

http://www.hbnxb.net/CN/Y2026/V41/I1/22

图/表 8

表1 引物信息

Tab.1 Primer information

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
SmaⅠ-PA7-OsASL1.1-F	CCCGGGATGGCCGCCGCCACCTCCCAA	GFP融合载体构建
Spe Ⅰ-PA7-OsASL1.1-R	ACTAGTTGAACTAATGCCAAGCTGAGT
qRT-PCR-OsASL1.1-F	CTTGTGAAGAAGGGCGTGC	实时荧光定量PCR
qRT-PCR-OsASL1.1-R	GCGTTCTCCACTCCCAGATA
qRT-PCR-ACTIN-F	CAACACCCCTGCTATGTACG	内参基因
qRT-PCR-ACTIN-R	CATCACCAGAGTCCAACACAA
Cas9-OsASL1.1-F	TGTGTCTCCCAATCCTTCCTCTCGC	Cas9-OsASL1.1
Cas9-OsASL1.1-R	AAACGCGAGAGGAAGGATTGGGAG	突变体构建
OsASL1.1-JD-F	GAGGACACACACACTCACCG	Cas9-OsASL1.1
OsASL1.1-JD-R	GAGGCAGGTGTTTCATCAAGC	突变体鉴定

图1 OsASL1.1蛋白的亚细胞定位(标尺=5 μm)

Fig.1 Subcellular localization of OsASL1.1 protein(Bars=5 μm)

表2 OsASL1.1基因启动子区顺式作用元件分析

Tab.2 Analysis of cis-acting element in OsASL1.1 promoter region

元件名称 Element name	功能 Function	数量/个 Number	基序(5'—3') Motif(5'—3')
GC-motif	参与缺氧特异性诱导的增强子元件	1	CCCCCG
CAT-box	与分生组织表达相关的顺式作用调控元件	1	GCCACT
LTR	参与低温响应的顺式作用元件	1	CCGAAA
ABRE	参与ABA响应的顺式作用调控元件	5	ACGTG/GCAACGTGTC/ GCCGCGTGGC/CACGTG/ACGTG
AuxRR-core	参与生长素响应的顺式作用调控元件	1	AACGAC
ARE	厌氧诱导所必需的顺式作用调控元件	1	AAACCA
CAAT-box	启动子与增强子区域常见元件	14	CAAT
TATA-box	核心启动子元件	17	TATA

图2 OsASL1.1基因启动子区ABRE的位置 UTR.非翻译区。图4同。

Fig.2 Position of ABA-responsive cis-acting elements(ABRE)in the promoter region of OsASL1.1 gene UTR.Untranslated region.The same as Fig.4.

图3 OsASL1.1基因在非生物胁迫下的表达模式不同小写字母表示不同处理间差异显著(P<0.05)。图5,6同。

Fig.3 Expression patterns of OsASL1.1 gene under abiotic stress Different lowercase letters mean significant difference among different treatments(P<0.05).The same as Fig.5,6.

图4 Cas9-OsASL1.1突变体的突变类型分析 A.Cas9-OsASL1.1突变体幼苗的PCR鉴定:1—22.突变体幼苗;23.阳性对照(Kitaake);24.阴性对照(无核酸酶无菌水)。B.Cas9-OsASL1.1突变体的突变类型分析。

Fig.4 Analysis of mutation types of Cas9-OsASL1.1 mutant A.PCR identification of Cas9-OsASL1.1 mutant seedlings:1—22.Mutant seedlings;23.Positive control(Kitaake);24.Negative control(nuclease-free sterile water).B.Analysis of mutation type of Cas9-OsASL1.1 mutant.

图5 ABA对野生型和Cas9-OsASL1.1突变体主根长的影响(标尺=2 cm) A.ABA处理下野生型和Cas9-OsASL1.1突变体的整体表型;B.ABA处理下野生型和Cas9-OsASL1.1突变体的单株表型;C.ABA处理下野生型和Cas9-OsASL1.1突变体的主根长分析;D.ABA处理下野生型和Cas9-OsASL1.1突变体的主根长抑制率分析。

Fig.5 Effect of ABA on primary root length of wild-type and Cas9-OsASL1.1 lines(Bars=2 cm) A.Overall phenotype of wild type and Cas9-OsASL1.1 lines under ABA treatment;B. Individual plant phenotype of wild type and Cas9-OsASL1.1 lines under ABA treatment;C.Primary root length analysis of wild type and Cas9-OsASL1.1 lines under ABA treatment;D.Inhibition rate analysis of primary root length of wild type and Cas9-OsASL1.1 lines under ABA treatment.

图6 甘露醇对野生型和Cas9-OsASL1.1突变体存活率的影响(标尺=2 cm) A.甘露醇处理下野生型和Cas9-OsASL1.1突变体的表型分析;B.甘露醇处理下野生型和Cas9-OsASL1.1突变体的存活率分析。

Fig.6 Effect of mannitol on survival rate of wild type and Cas9-OsASL1.1 lines(Bars=2 cm) A.Analysis of phenotypic of wild type and Cas9-OsASL1.1 lines under mannitol treatment;B.Analysis of survival rate of wild type and Cas9-OsASL1.1 lines under mannitol treatment.

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基于CRISPR/Cas9技术的水稻OsASL1.1基因突变体创制及干旱胁迫下的表型分析

Creation of Rice OsASL1.1 Mutants Based on CRISPR/Cas9 Technology and Phenotypic Analysis under Drought Stress

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基于CRISPR/Cas9技术的水稻OsASL1.1基因突变体创制及干旱胁迫下的表型分析

Creation of Rice OsASL1.1 Mutants Based on CRISPR/Cas9 Technology and Phenotypic Analysis under Drought Stress

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