Identification of Knockout of BRI1 Mutant in Arabidopsis Mediated by CRISPR/Cas9

doi:10.7525/j.issn.1673-5102.2021.03.006

Abstract

Abstract:

CRISPR/Cas9 gene editing technology was used to specificity edit the BRI1 of Arabidopsis thaliana to creation new BRI1 mutants. We analyzed the sequence of BRI1 inthe obtained transgenic plants， the results showed that as one new BRI1 mutant due to the insertion of new bases， the normal BRI1 protein is terminated prematurely. This mutant have the similar phenotype as bri1-710， and both mutants show impaired response to BL treatment compared with wild-type. Those results suggested that the N-terminal of BRI1 may play a role in the BR signaling pathway. Therefore， this study could provide a reliable reference for further studies on the BRI1 function of A. thaliana and other homologous species.

Key words: CRISPR/Cas9, gene knockout, Arabidopsis, BRI1.

CLC Number:

S759.95

Guo-Fan WU, Hong-Bin CHENG, Yu-Jun WU, Juan SHEN, Wang-Ze WU. Identification of Knockout of BRI1 Mutant in Arabidopsis Mediated by CRISPR/Cas9[J]. Bulletin of Botanical Research, 2021, 41(3): 362-371.

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序列编号 SeqID	NGG错配碱基数 minMM_GG	NAG错配碱基数 minMM_AG	间隔序列 Spacer seq(5′-3′)	PAM序列 PAM(5′-3′)	正反链 Strand	位置 Location
Chr4:18324967-18324987	3	3	ACAAGAATCTTCTCCCAGAC	TGGTCTTCCA	+	exon
Chr4:18325007-18325027:c	5	5	TAACGCCATCGAAAGTACAC	GGGTTTTTGT	-	exon
Chr4:18325008-18325028:c	4	4	GTAACGCCATCGAAAGTACA	CGGGTTTTTG	-	exon
Chr4:18325069-18325089:c	3	3	GAATCCGACGTTGAGAGGCT	TGGAGCTGAG	-	exon
Chr4:18325062-18325082	3	4	AGCTCCAAGCCTCTCAACGT	CGGATTCAGT	+	exon
Chr4:18325112-18325132:c	3	3	ACTCTAATCCGGTGAGAGAC	AGGAGAGACG	-	exon
Chr4:18325152-18325172	4	4	CACATCAATGGCTCCGTTTC	TGGCTTCAAG	+	exon
Chr4:18325168-18325188:c	4	4	AGAGCACTTGAAGCCAGAAA	CGGAGCCATT	-	exon
Chr4:18325201-18325221:c	3	3	GTTTCTAGATAGATCCAAGC	TGGTTAAAGA	-	exon
Chr4:18325236-18325256	4	4	GTAACGACTCTAACAAGCCT	TGGTTCTTGC	+	exon
Chr4:18325256-18325276:c	5	5	TCAGACCGGAGCAAGAACCA	AGGCTTGTTA	-	exon
Chr4:18325248-18325268	4	4	ACAAGCCTTGGTTCTTGCTC	CGGTCTGAAG	+	exon

组成成分 Composition ingredient	体积 Volume（μL）	反应条件 Reaction condition
PCR片段（626 bp）	2	5 hours at 37℃ 5 min at 50℃ 10 min at 80℃
Phee401	2
10×NEB T4 Buffer	1.5
10×BSA	1.5
Bsal（NEB）	1
T4 Ligase（NEB）	1
ddH₂O	6
总计Total	15	体系

引物名称 Primer name	引物序列 Primer sequence（5′-3′）	用途 Usage
DT1-BsF	ATATATGGTCTCGATTGGCTCCAAGCCTCTCAACGTGTT	载体构建 Vector construction
DT1-F0	TGGCTCCAAGCCTCTCAACGTGTTTTAGAGCTAGAAATAGC
DT2-R0	AACACGTTGAGAGGCTTGGAGCCAATCTCTTAGTCGACTCTAC
DT2-BsR	ATTATTGGTCTCGAAACACGTTGAGAGGCTTGGAGCC
U626-IDF	TGTCCCAGGATTAGAATGATTAGGC	载体鉴定 Vector identification
U629-IDR	AGCCCTCTTCTTTCGATCCATCAAC	载体鉴定 Vector identification
AtBRI1-F	TGAGAGACAGGAGAGACGAG	阳性植株检测 Detection of positive plant
AtBRI1-R	CAAGCTTCACCATCTCAGTC	阳性植株检测 Detection of positive plant
ACT2-Q-F	TGTGCCAATCTACGAGGGTTT	荧光定量PCR分析 qRT-PCR analysis
ACT2-Q-R	TTTCCCGCTCTGCTGTTGT
CPD-F	GAGACGCTACGAGTGGCTAA
CPD-R	GCATCTTTGAAGTGGTTTGGG
DWF4-F	CCACAACACTCGGTGACTTC
DWF4-R	CAGCTGATACGATCGTTGGTT
BR6ox2-F	CGGTTACCCGCAATCTATG
BR6ox2-R	TAAAGAAAGCAACGAGCCTC
SAUR-AC1-F	AGATATGTGGTGCCGGTTTC
SAUR-AC1-R	TTGTTAAGCCGCCCATTG

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