利用CRISPR/Cas9技术编辑水稻香味基因Badh2

doi:10.3864/j.issn.0578-1752.2020.08.001

Abstract

Abstract:

【Objective】Rice fragrance, a very important trait for quality improvement, is mainly controlled by a recessive gene Badh2. In this study, CRISPR/Cas9-mediated editing were used to generate the gene-edited rice plants with the fragrant Badh2 in the conventional elite rice varieties, and the trait of fragrance was improved.【Method】 The targeted sequences were designed according to the sequence of exon2 and exon7 of the Badh2 by the principle of CRISPR/Cas9 gene editing. Its specificity of the targeted sequence was determined by BLAST analysis, and then constructed into CRISPR/Cas9 expression vector. The callus of Jia58 and Xiushui134 which are widely cultivated in the Zhejiang Province were selected as explants to transform by Agrobacterium-mediated genetic transformation, and the positive transgenic plants were obtained by the screening of hygromycin resistance. Sequencing analysis of transgenic lines was used to detect the presence of the mutation type on the loci of Badh2. Stable marker-free gene-edited lines carrying the mutation on Badh2 were obtained by PCR analysis and identification. The content of 2-AP in the brown rice flour were measured by GC-MS, and the difference between the gene-edited lines and non-transgenic control was determined. 【Result】 Badh2 of the transgenic lines was directionally mutated by the genetic transformation using the expression vector on which the target sequence of the exon2 and exon7 were designed and constructed. A total of 15 T₀ gene-edited lines were obtained from Jia58. Eight of them were generated mutation on the exon2 with five different mutations types in which different single base was inserted into different position. Seven of them were generated mutations on the exon7 with five different mutation types in which the base or fragment deletion was produced. A total of 11 T₀ gene-edited lines were obtained from Xiushui134, of which five lines were generated mutations on the exon2 with the single base insertion and six lines on the exon7 with the fragment deletion. A total of 16 marker-free gene-edited lines were obtained from 48 T₁ Xiushui134, of which five lines were generated mutations on the exon2, and 11 lines on the exon7. The average 2-AP content in brown rice flour of four T₂gene-edited lines were 0.309, 0.347, 0.332 and 0.295 μg·g ^-1 respectively, which were significantly higher (P<0.01) than that of the non-transgenic control (0.046 μg·g ^-1). 【Conclusion】 The Badh2 which controlled the rice fragrance trait was directionally edited by using CRISPR/Cas9-mediated technology, and the marker-free gene-edited lines were obtained, of which the fragrance of Badh2 edited lines were significantly improved.

Key words: rice, CRISPR/Cas9, gene editing, Badh2, fragrance

QI YongBin,ZHANG LiXia,WANG LinYou,SONG Jian,WANG JianJun. CRISPR/Cas9 Targeted Editing for the Fragrant Gene Badh2 in Rice[J].Scientia Agricultura Sinica, 2020, 53(8): 1501-1509.

Figures/Tables 5

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References 32

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引物 Primers	引物序列Primer sequence(5′-3′)	用途Usage
SG-SR3	GGCCATTTGTCTGCAGAAT	鉴定载体 Vector verification
PUV4-R	TCCCAGTCACGACGTTGTAA	鉴定载体 Vector verification
Cas9-F	CATCCAGAAAGCCCAGGTGT	鉴定Cas9 PCR Detection for Cas9
Cas9-R	GTTCCTGGTCCACGTACATA	鉴定Cas9 PCR Detection for Cas9
Hpt-F	GGGTGTCACGTTGCAAGACC	鉴定Hpt PCR Detection for Hpt
Hpt-R	ATGCCTCCGCTCGAAGTAGC	鉴定Hpt PCR Detection for Hpt
Oligo-up1	TGTGTGGCGATTGCGCGGAGGTACT	构建Exon2靶点二聚体Construct target Oligo for Exon2
Oligo-lw1	AAACAGTACCTCCGCGCAATCGCCA	构建Exon2靶点二聚体Construct target Oligo for Exon2
Oligo-up2	TGTGTGATGGCTTCAGCTGCTCCTA	构建Exon7靶点二聚体Construct target Oligo for Exon7
Oligo-lw2	AAACTAGGAGCAGCTGAAGCCATCA	构建Exon7靶点二聚体Construct target Oligo for Exon7
exon2-F	CACCCTCTGCTTCTGCCTCT	Exon2测序 Sequencing for Exon2
exon2-R	CAGCCATGCTTCCAACTTATTC	Exon2测序 Sequencing for Exon2
exon7-F	TGGTCTTCCTTCAGGTGTGC	Exon7测序 Sequencing for Exon7
exon7-R	TCCAGTGAAACAGGCTGTCA	Exon7测序 Sequencing for Exon7

CRISPR/Cas9 Targeted Editing for the Fragrant Gene Badh2 in Rice

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