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

doi:10.7668/hbnxb.20195973

Abstract

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

CLC Number:

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.

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Figures/Tables 8

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

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

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

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.

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.

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.

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.

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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