CRISPR/Cas9 Targeted Editing for the Fragrant Gene Badh2 in Rice
QI YongBin, ZHANG LiXia, WANG LinYou, SONG Jian, WANG JianJun,Institute of Crop Science and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021
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 T2 gene-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. Keywords:rice;CRISPR/Cas9;gene editing;Badh2;fragrance
PDF (2664KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文 本文引用格式 祁永斌, 张礼霞, 王林友, 宋建, 王建军. 利用CRISPR/Cas9技术编辑水稻香味基因Badh2[J]. 中国农业科学, 2020, 53(8): 1501-1509 doi:10.3864/j.issn.0578-1752.2020.08.001 QI YongBin, ZHANG LiXia, WANG LinYou, SONG Jian, WANG JianJun. CRISPR/Cas9 Targeted Editing for the Fragrant Gene Badh2 in Rice[J]. Scientia Acricultura Sinica, 2020, 53(8): 1501-1509 doi:10.3864/j.issn.0578-1752.2020.08.001
LB:载体左边界;U6:水稻OsU6启动子;SG:向导RNA;UBI:UBI启动子;Cas9:Cas9蛋白;Nos Ter:Nos终止子;35S:35S启动子;Hygro:潮霉素基因;Poly A Ter:PolyA终止子;RB:载体右边界 Fig. 1Diagram of the map of CRISPR/Cas9
LB: Left border; U6: OsU6 promoter; SG: sgRNA; UBI: UBI promoter; Cas9: Cas9 protein; Nos Ter: Nos terminator; 35S: 35S promoter; Hygro: Hygromycin; Poly A Ter: Poly A terminator; RB: Right border
Start code:起始密码子;Stop code:终止密码子;Exon2:第2外显子;Exon7:第7外显子;sgRNA target site:sgRNA靶点。方框表示靶标序列,红色下划线字体表示PAM序列,蓝色字体表示插入碱基,横线表示缺失碱基,+表示碱基插入,-表示碱基缺失 Fig. 2Diagram of the sgRNA design and mutation analysis on the exon2 and exon7 of Badh2 gene
The box represented target sequence. The red words with the underline represented PAM sequence. The blue words indicated the insertion base. The transverse line indicated the deletion sequences. + represented base insertion, -indicated base deletion
LI QL, GAO XR, YU XH, WANG XZ, AN LJ . Molecular cloning and characterization of betaine aldehyde dehydrogenase gene from Suaeda liaotungensis and its use in improved tolerance to salinity in transgenic tobacco , 2003,25(17):1431-1436. [本文引用: 1]
CHENS, YANGY, SHIW, JIQ, HEF, ZHANGZ, CHENGZ, LIUX, XUM . Badh2, encoding betaine aldehyde dehydrogenase, inhibits the biosynthesis of 2-acetyl-1-pyrroline, a major component in rice fragrance , 2008,20(7):1850-1861. [本文引用: 2]
BRADBURY LM, FITZGERALD TL, HENRY RJ, JINQ, WATERS DL . The gene for fragrance in rice , 2005,3(3):363-370. [本文引用: 1]
SAKTHIVELK, SUNDARAM RM, SHOBHA RANIN, BALACHANDRAN SM, NEERAJA CN . Genetic and molecular basis of fragrance in rice , 2009,27(4):468-473. [本文引用: 1]
OKPALA NE, MOZ, DUANM, TANGX . The genetics and biosynthesis of 2-acetyl-1-pyrroline in fragrant rice , 2019,135:272-276. [本文引用: 1]
YU YY, SHAO GN, SHENG ZH, JIANG HW, HE JW, SUN YY, CAI YC, HU PS, TANG SQ . Genetic diversity of global aromatic rice varieties Plant Diversity and Resources, 2015,37(6):871-880. (in Chinese) [本文引用: 1]
SHAOG, TANGS, CHENM, WEIX, HEJ, LUOJ, JIAOG, HUY, XIEL, HUP . Haplotype variation at Badh2, the gene determining fragrance in rice , 2013,101(2):157-162. [本文引用: 1]
ZHANG JL, LI SJ, LIJ, PU SH, PU YJ, ZHANGL, TAN YL, CHEN LJ, TAN XL, JIN SL, WEN JC . Identification of the fragrant gene badh2 locus in rice germplasm resources original from different area Molecular Plant Breeding, 2015,13(4):727-733. (in Chinese) [本文引用: 1]
SOOD BC, SIDDIQ EA . A rapid technique for scent determination in rice , 38(1978):268-271. [本文引用: 1]
LORIEUXM, PETROVM, HUANGN, GUIDERDONIE, GHESQUIèREA . Aroma in rice: genetic analysis of a quantitative trait , 1996,93(7):1145-1151. [本文引用: 1]
JIN QS, WATERSD, CORDEIRO GM, HENRY RJ, REINKE RF . A single nucleotide polymorphism (SNP) marker linked to the fragrance gene in rice ( Oryza sativa L.) , 2003,165:359-364. [本文引用: 2]
MASOULEH AK, WATERS DL, REINKE RF, HENRY RJ . A high-throughput assay for rapid and simultaneous analysis of perfect markers for important quality and agronomic traits in rice using multiplexed MALDI-TOF mass spectrometry , 2009,7(4):355-363. [本文引用: 2]
LU YT, LIU QL, WANG JM, YAN WC, YU FM, JIN QS . Detection of rice fragrant gene by allele-specific amplification Acta Agronomica Sinica, 2008,34(2):243-246. (in Chinese) [本文引用: 2]
NIUX, TANGW, HUANGW, RENG, WANGQ, LUOD, XIAOY, YANGS, WANGF, LUBR, GAOF, LUT, LIUY . RNAi-directed downregulation of OsBADH2 results in aroma (2-acetyl-1-pyrroline) production in rice (Oryza sativa L.) , 2008,8:100. [本文引用: 2]
CHEN ML, WEI XJ, SHAO GN, TANG SQ, LUOJ, HU PS . Fragrance of the rice grain achieved via artificial microRNA-induced down-regulation of OsBADH2 , 2012,131:584-590. [本文引用: 1]
SHANQ, WANGY, CHENK, LIANGZ, LIJ, ZHANGY, ZHANGK, LIUJ, VOYTAS DF, ZHENGX, ZHANGY, GAOC . Rapid and efficient gene modification in rice and Brachypodium using TALENs , 2013,6:1365-1368. [本文引用: 1]
CANTOSC, FRANCISCOP, TRIJATMIKO KR, SLAMET-LOEDINI, CHADHA-MOHANTY PK . Identification of "safe harbor" loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair , 2014, 26; 5:302. [本文引用: 1]
LIANGZ, ZHANGK, CHENK, GAOC . Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system , 2014,41:63-68. [本文引用: 1]
SHANQ, ZHANGY, CHENK, ZHANGK, GAOC . Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology , 2015,13(6):791-800. [本文引用: 2]
SHAO GN, XIE LH, JIAO GA, WEI XJ, SHENG ZH, TANG SQ, HU PS . CRISPR/CAS9-mediated editing of the fragrant gene Badh2 in rice Chinese Journal of Rice Science, 2017,31(2):216-222. (in Chinese) [本文引用: 2]
JAINS, JAIN RK, MCCOUCH SR . Genetic analysis of Indian aromatic and quality rice ( Oryza sativa L.) germplasm using panels of fluorescently-labeled microsatellite markers , 2004,109(5):965-977. [本文引用: 1]
PACHAURIV, SINGH MK, SINGH AK . Origin and genetic diversity of aromatic rice varieties,molecular breeding and chemical and genetic basis of rice aroma , 2010,19(2):127-143. [本文引用: 1]
ZHENG JT, YANG DW, DONG LF, YOU QR, ZHENGY, TU SH, ZHOUP . Inheritance and breeding actuality of new quasi-aromatic rice ( Oryza sativa L.) Fujian Journal of Agricultural Sciences, 2012,27(10):1134-1138. (in Chinese) [本文引用: 1]
HUANG TX, JIANG WQ, YOU QR, ZHOU SQ, LIU DH, XIE DR, QIU HM . Breeding and utilization of fragrant restoring line Dalixiang 15 of indica hybrid rice Fujian Journal of Agricultural Science, 2006,21(2):83-88. (in Chinese) [本文引用: 1]
FRIEDLAND AE, TZUR YB, ESVELT KM, COLAIáCOVO MP, CHURCH GM, CALARCO JA . Heritable genome editing in C. elegans via a CRISPR-Cas9 system , 2013,10:741-743. [本文引用: 1]
XIEK, ZHANGJ, YANGY . Genome-wide prediction of highly specific guide RNA spacers for CRISPR-Cas9-mediated genome editing in model plants and major crops , 2014,7:923-926. [本文引用: 1]
FENGZ, MAOY, XUN, ZHANGB, WEIP, YANG DL, WANGZ, ZHANGZ, ZHENGR, YANGL, ZENGL, LIUX, ZHU JK . Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis , 2014,111:4632-4637. [本文引用: 1]
LUOM, GILBERTB, AYLIFFEM . Applications of CRISPR/Cas9 technology for targeted mutagenesis, gene replacement and stacking of genes in higher plants , 2016,35(7):1439-1450. [本文引用: 1]
ENDOM, MIKAMIM, TOKIS . Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice , 2015,56:41-47. [本文引用: 1]
PATTANAYAKV, LINS, GUILINGER JP, MAE, DOUDNA JA, LIU DR . High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity , 2013,31(9):839-843. [本文引用: 1]