基于CRISPR/Cas9技术的水稻OsDUF1475突变体的创建与分析
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摘要
未知功能结构域蛋白家族(domains of unknown function protein families, DUFs)是一大群功能未表征的蛋白家族,在生命活动中具有重要作用。为了探究水稻(Oryza sativa) DUF基因OsDUF1475的生物学功能,利用CRISPR/Cas9技术对OsDUF1475基因进行靶向编辑。针对OsDUF1475基因第1个外显子设计2个single guide RNA (sgRNA)靶位点,分别构建获得2个CRISPR/Cas9重组载体;然后通过农杆菌(Agrobacterium)介导的遗传转化法导入水稻品种日本晴(Nipponbare)中,经潮霉素抗性筛选之后获得65株T0代转基因植株。对转基因植株的靶位点进行测序分析,发现有单碱基插入、10 bp以内碱基缺失和大片段DNA序列缺失3种类型。进一步分析氨基酸序列,发现这些突变造成氨基酸移码现象,进而导致蛋白质翻译的提前终止。上述结果表明,CRISPR/Cas9重组载体成功实现了对OsDUF1475基因的定向编辑,并且通过自交获得了T1代纯合突变植株。这些突变植株为进一步开展OsDUF1475功能研究提供了基础资料。
Domains of unknown function protein families(DUFs) are a large set of uncharacterized protein families, which play an important role in biological activities. To investigate biological function of the rice(Oryza sativa) DUF gene OsDUF1475, the CRISPR/Cas9 gene editing technique was employed. Two target sites on the first exon of the OsDUF1475 gene were selected and ligased into CRISPR/Cas9 vector to produce2 recombinant vectors. Then, the 2 recombined plasmids were introduced into rice cultivar Nipponbare by Agrobacterium-mediated transformation method. Totally 65 T0 transgenic plants were obtained. Sequencing analysis of the target sites showed that there were 3 types of mutations in the transgenic plants, including single base insertions, short fragment deletions(<10 bp) and long fragment deletions(>10 bp). Analysis of the amino acid sequence showed that the frame shift of OsDUF1475 ORF were produced which resulted intruncated protein in editing mutants. The results indicated that the OsDUF1475 gene was edited successfully by CRISPR/Cas9 technique. And T1 generation homozygous mutant plants obtained by self-crossing showed that these mutants were genetically stable. Taken together, the mutants obtained in present study are suitable materials for OsDUF1475 function study.
Domains of unknown function protein families(DUFs) are a large set of uncharacterized protein families, which play an important role in biological activities. To investigate biological function of the rice(Oryza sativa) DUF gene OsDUF1475, the CRISPR/Cas9 gene editing technique was employed. Two target sites on the first exon of the OsDUF1475 gene were selected and ligased into CRISPR/Cas9 vector to produce2 recombinant vectors. Then, the 2 recombined plasmids were introduced into rice cultivar Nipponbare by Agrobacterium-mediated transformation method. Totally 65 T0 transgenic plants were obtained. Sequencing analysis of the target sites showed that there were 3 types of mutations in the transgenic plants, including single base insertions, short fragment deletions(<10 bp) and long fragment deletions(>10 bp). Analysis of the amino acid sequence showed that the frame shift of OsDUF1475 ORF were produced which resulted intruncated protein in editing mutants. The results indicated that the OsDUF1475 gene was edited successfully by CRISPR/Cas9 technique. And T1 generation homozygous mutant plants obtained by self-crossing showed that these mutants were genetically stable. Taken together, the mutants obtained in present study are suitable materials for OsDUF1475 function study.
引文
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Kim S J,Ryu M Y,Kim W T.2012.Suppression of Arabidopsis,RING-DUF1117 E3 ubiquitin ligases,AtRDUF1,and AtRDUF2,reduces tolerance to ABA-mediated drought stress[J].Biochemical&Biophysical Research Communications,420(1):141-147.
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Zhang H,Zhang J,Wei P,et al.2014.The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation[J].Plant Biotechnology Journal,12(6):797-807.
Zhou H,Liu B,Weeks D P,et al.2014.Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice[J].Nucleic Acids Research,42(17):10903-10914.
刘志国.2014.CRISPR/Cas9系统介导基因组编辑的研究进展[J].畜牧兽医学报,45(10):1567-1583.(Liu Z G2014.Research progress on CRISPR/Cas9 mediated genome editing[J].Chinese Journal of Animal&Veterinary Sciences,45(10):1567-1583.)
秦丹丹,谢颂朝,刘刚,等.2013.小麦中编码未知蛋白的热胁迫响应基因TaWTF1的克隆和功能分析[J].植物学报,48(1):34-41.(Qin D D,Xie S C,Liu G,et al.2013Isolation and functional characterization of heat-stressresponsive gene TaWTF1 from wheat[J].Chinese Bulletin of Botany,48(1):34-41.)
Bedell V M,Wang Y,Campbell J M,et al.2012.In vivo genome editing using a high-efficiency TALEN system[J]Nature,491(7422):114-118.
Bibikova M,Beumer K,Trautman J K,et al.2003.Enhancing gene targeting with designed zinc finger nucleases[J]Science,300(5620):764-764.
Bischoff V,Selbig J,Scheible W R.2010.Involvement of TBL/DUF231 proteins into cell wall biology[J].Plan Signaling&Behavior,5(8):1057-1059.
Cong L,Ran F A,Cox D,et al.2013.Multiplex genome engineering using CRISPR/Cas systems[J].Science,32(12):819-823.
Hiei Y,Ohta S,Komari T,et al.1994.Efficient transformation of rice(Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA[J].Plant Journal,6(2):271-282.
Hsu P D,Lander E S,Zhang F.2014.Development and applications of CRISPR-Cas9 for genome engineering[J].Cell,157(6):1262-1278.
Jinek M,Chylinski K,Fonfara I,et al.2012.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science,337(6096):816-821.
Kim S,Kang Y Y,Wu J,et al.2010.Secretome analysis of differentially induced proteins in rice suspension-cultured cells triggered by rice blast fungus and elicitor[J].Proteomics,9(5):1302-1313.
Kim S J,Ryu M Y,Kim W T.2012.Suppression of Arabidopsis,RING-DUF1117 E3 ubiquitin ligases,AtRDUF1,and AtRDUF2,reduces tolerance to ABA-mediated drought stress[J].Biochemical&Biophysical Research Communications,420(1):141-147.
Mudgal R,Sandhya S,Chandra N,et al.2015.De-DUFing the DUFs:Deciphering distant evolutionary relationships of Domains of Unknown Function using sensitive homology detection methods[J].Biology Direct,10(1):38.
Schultz J,Milpetz F,Bork P,et al.1998.SMART,a simple modular architecture research tool:Identification of signaling domains[J].Proceedings of the National Academy of Sciences of the USA,95(11):5857-5864.
Yan D,Zhou Y,Ye S,et al.2013.Beak-shaped grain 1/TRI-ANGULAR HULL 1,a DUF640 gene,is associated with grain shape,size and weight in rice[J].Science China Life Sciences,56(3):275-283.
Zhang H,Zhang J,Wei P,et al.2014.The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation[J].Plant Biotechnology Journal,12(6):797-807.
Zhou H,Liu B,Weeks D P,et al.2014.Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice[J].Nucleic Acids Research,42(17):10903-10914.