水稻OsDUF393基因编辑突变体的创建
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摘要
DUFs家族是一类未知功能结构域蛋白家族,转录谱和蛋白谱分析发现,DUFs蛋白在生物的生长和发育中发挥着至关重要的作用,因此对DUFs基因功能的研究将有助于了解生物体复杂的生长发育机制。目前该家族基因功能仍鲜见报道。为了探究水稻(Oryza sativa)OsDUF393(Domains of Unknown Function 393)基因的生物学功能,利用CRISPR/Cas9技术构建了水稻DUFs家族基因OsDUF393的载体,同时利用农杆菌介导的遗传转化将构建的CRISPR/Cas9载体转化水稻品种中花11中,经潮霉素抗性基因鉴定,获得了100株T0代转基因植株。对转基因植株的靶位点进行测序分析后发现有单碱基插入和大片段DNA序列缺失2种类型。通过RT-PCR分析突变体中OsDUF393基因的表达水平,结果表明,目的基因在不同纯合编辑突变体中转录水平均有下降。上述结果表明,CRISPR/Cas9重组载体成功实现了对OsDUF393基因的定向编辑。这些编辑突变体材料为后续该基因功能研究奠定了基础。
DUFs protein families are a large of uncharacterized protein families with unknown functional domain. Transcription profile and protein profile revealed that DUF protein play a crucial role in the biological process of development and growth. Therefore, the study of DUFs gene function will be helpful to understand the complicated mechanism of growth and development of organisms. To investigate biological function of the rice gene OsDUF393(Domains of Unknown Function 393), the CRISPR/Cas9 gene editing technique was employed. In this study, we constructed a CRISPR/Cas9 vector editing OsDUF393 of rice and introduced it into rice variety Zhonghua11 to produce transgenic plants by Agrobacterium-mediated plant transformation method. Totally 100 T0 transgenic plants were obtained. Sequencing analysis of the target sites showed that there are two types of mutations in the transgenic plants, including single base insertions and long fragment deletions. The expression level of OsDUF393 gene in the mutant was analyzed by RT-PCR,and the results showed that the transcription level of the target gene was decreased in different homozygous editing mutants. The results indicated that the OsDUF393 gene was edited successfully by CRISPR/Cas9 system. Taken together, these mutants are suitable materials for OsDUF393 function study.
DUFs protein families are a large of uncharacterized protein families with unknown functional domain. Transcription profile and protein profile revealed that DUF protein play a crucial role in the biological process of development and growth. Therefore, the study of DUFs gene function will be helpful to understand the complicated mechanism of growth and development of organisms. To investigate biological function of the rice gene OsDUF393(Domains of Unknown Function 393), the CRISPR/Cas9 gene editing technique was employed. In this study, we constructed a CRISPR/Cas9 vector editing OsDUF393 of rice and introduced it into rice variety Zhonghua11 to produce transgenic plants by Agrobacterium-mediated plant transformation method. Totally 100 T0 transgenic plants were obtained. Sequencing analysis of the target sites showed that there are two types of mutations in the transgenic plants, including single base insertions and long fragment deletions. The expression level of OsDUF393 gene in the mutant was analyzed by RT-PCR,and the results showed that the transcription level of the target gene was decreased in different homozygous editing mutants. The results indicated that the OsDUF393 gene was edited successfully by CRISPR/Cas9 system. Taken together, these mutants are suitable materials for OsDUF393 function study.
引文
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[3]陈艳红,杜菊萍,刘建胜,等. DUF784基因在花粉管导向中的功能分析[J].中国生物化学与分子生物学报,2010,26(10):903-910.
[4] JENSEN J K, KIM H, COCURON J C, et al. The DUF579 domain containing proteins IRX15 and IRX15‐L affect xylan synthesis in Arabidopsis[J]. Plant J, 2011, 66(3):387-400.
[5] GAO Y, BADEJO A A, SAWA Y, et al. Analysis of two LGalactono-1,4-lactone-responsive genes with complementary expression during the development of Arabidopsis thaliana[J]. Plant Cell Physiol, 2012, 53(3):592-601.
[6] ZHANG H, ZHANG T T, LIU H, et al. Thioredoxin-mediated ROS homeostasis explains natural variation in plant regeneration[J]. Plant Physiol, 2017, 7(1):2 231-2 250.
[7] LUO C, GUO C, WANG W, et al. Overexpression of a new stress-repressive gene OsDSR2 encoding a protein with a DUF966 domain increases salt and simulated drought stress sensitivities and reduces ABA sensitivity in rice[J]. Plant Cell Rep, 2014, 33(2):323-336.
[8]王艳杰. DUF1644家族基因OsSIDP409在水稻盐胁迫应答中的功能分析[D].厦门:厦门大学,2014.
[9] YAN D, ZHOU Y, YE S, et al. Beak-shaped grain 1/TRIANGULAR HULL 1, a DUF640 gene, is associated with grain shape, size and weight in rice[J]. Sci China Life Sci, 2013, 56(3):275-283.
[10] ISHINO Y, SHINAGAWA H, MAKINO K, et al. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product[J]. J Bacteriol, 1987, 169(12):5 429-5 433.
[11] JANSEN R, EMBDEN J D, GAASTRA W, et al. Identification of genes that are associated with DNA repeats in prokaryotes[J]. Mol Microbiol, 2002, 43(6):1 565-1 575.
[12] BARRANGOU R, FREMAUX C, DEVEAU H, et al. CRISPR provides acquired resistance against viruses in prokaryotes[J]. Science, 2007,315(5819):1 709-1 712.
[13] BHAYA D, DAVISON M, BARRANGOU R. CRISPR-Cas systems in bacteria and archaea:versatile small RNAs for adaptive defense and regulation[J]. Annu Rev Genet, 2011, 45(45):273-97.
[14] CONG L, RAN F A, COX D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 32(12):819-823.
[15] BELHAJ K, CHAPARRO-GARCIA A, KAMOUN S, et al. Editing plant genomes with CRISPR/Cas9[J]. Curr Opin Biotechnol, 2015,32:76-84.
[16] LI J, ZHANG Y, CHEN K, et al. Targeted genome modification of crop plants using a CRISPR-Cas system[J]. Nat Biotechnol, 2013, 31(8):686-688.
[17] JIN M, GUO D, ZHANG J, et al. Targeted mutagenesis in rice using CRISPR-Cas system[J]. Cell Res, 2013, 23(10):1 233-1 236.
[18] FENG Z, ZHANG B, DING W, et al. Efficient genome editing in plants using a CRISPR/Cas system.[J]. Cell Res, 2013, 23(10):1 229-1 232.
[19] ZHANG J, ZHANG H, BOTELLA J R, et al. Generation of new glutinous rice by CRISPR/Cas9‐targeted mutagenesis of the Waxy gene in elite rice varieties[J]. J Integr Plant Biol, 2018, 60(5):369-375.
[20] WANG M, MAO Y, LU Y, et al. Multiplex gene editing in rice using the CRISPR-Cpf1 system[J]. Mol Plant, 2017, 10(7):1 011-1 013.
[21] HIEI Y, OHTA S, KOMARI T, et al. Efficient transformation of rice(Oryza sativa L.)mediated by agrobacterium and sequence analysis of the boundaries of the T-DNA[J]. Plant J,1994, 6(2):271–282.
[22] ZHANG G H, GAO M G, ZHANG G Z, et al. A high through-put protocol of plant genomic DNA Preparation for PCR[J]. Acta Agronomica Sinica, 2013, 39(7):1 200.
[23] XUE H Y, JI L J, GAO A M, et al. CRISPR-Cas9 for medical genetic screens:applications and future perspectives[J]. J Med Genet, 2016,53(2):91.
[24]李娟,常闪闪,刘凤权,等. Os DUF500基因沉默提高水稻对白叶枯病的抗性[J].中国水稻科学,2012,26(4):476-480.
[25]黄建平.水稻逆境诱导型启动子和低温应答基因Os LCL3的分离与鉴定[D].武汉:华中农业大学,2015.
[2]秦丹丹,谢颂朝,刘刚,等.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,2013,48(1):34-41.
[3]陈艳红,杜菊萍,刘建胜,等. DUF784基因在花粉管导向中的功能分析[J].中国生物化学与分子生物学报,2010,26(10):903-910.
[4] JENSEN J K, KIM H, COCURON J C, et al. The DUF579 domain containing proteins IRX15 and IRX15‐L affect xylan synthesis in Arabidopsis[J]. Plant J, 2011, 66(3):387-400.
[5] GAO Y, BADEJO A A, SAWA Y, et al. Analysis of two LGalactono-1,4-lactone-responsive genes with complementary expression during the development of Arabidopsis thaliana[J]. Plant Cell Physiol, 2012, 53(3):592-601.
[6] ZHANG H, ZHANG T T, LIU H, et al. Thioredoxin-mediated ROS homeostasis explains natural variation in plant regeneration[J]. Plant Physiol, 2017, 7(1):2 231-2 250.
[7] LUO C, GUO C, WANG W, et al. Overexpression of a new stress-repressive gene OsDSR2 encoding a protein with a DUF966 domain increases salt and simulated drought stress sensitivities and reduces ABA sensitivity in rice[J]. Plant Cell Rep, 2014, 33(2):323-336.
[8]王艳杰. DUF1644家族基因OsSIDP409在水稻盐胁迫应答中的功能分析[D].厦门:厦门大学,2014.
[9] YAN D, ZHOU Y, YE S, et al. Beak-shaped grain 1/TRIANGULAR HULL 1, a DUF640 gene, is associated with grain shape, size and weight in rice[J]. Sci China Life Sci, 2013, 56(3):275-283.
[10] ISHINO Y, SHINAGAWA H, MAKINO K, et al. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product[J]. J Bacteriol, 1987, 169(12):5 429-5 433.
[11] JANSEN R, EMBDEN J D, GAASTRA W, et al. Identification of genes that are associated with DNA repeats in prokaryotes[J]. Mol Microbiol, 2002, 43(6):1 565-1 575.
[12] BARRANGOU R, FREMAUX C, DEVEAU H, et al. CRISPR provides acquired resistance against viruses in prokaryotes[J]. Science, 2007,315(5819):1 709-1 712.
[13] BHAYA D, DAVISON M, BARRANGOU R. CRISPR-Cas systems in bacteria and archaea:versatile small RNAs for adaptive defense and regulation[J]. Annu Rev Genet, 2011, 45(45):273-97.
[14] CONG L, RAN F A, COX D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 32(12):819-823.
[15] BELHAJ K, CHAPARRO-GARCIA A, KAMOUN S, et al. Editing plant genomes with CRISPR/Cas9[J]. Curr Opin Biotechnol, 2015,32:76-84.
[16] LI J, ZHANG Y, CHEN K, et al. Targeted genome modification of crop plants using a CRISPR-Cas system[J]. Nat Biotechnol, 2013, 31(8):686-688.
[17] JIN M, GUO D, ZHANG J, et al. Targeted mutagenesis in rice using CRISPR-Cas system[J]. Cell Res, 2013, 23(10):1 233-1 236.
[18] FENG Z, ZHANG B, DING W, et al. Efficient genome editing in plants using a CRISPR/Cas system.[J]. Cell Res, 2013, 23(10):1 229-1 232.
[19] ZHANG J, ZHANG H, BOTELLA J R, et al. Generation of new glutinous rice by CRISPR/Cas9‐targeted mutagenesis of the Waxy gene in elite rice varieties[J]. J Integr Plant Biol, 2018, 60(5):369-375.
[20] WANG M, MAO Y, LU Y, et al. Multiplex gene editing in rice using the CRISPR-Cpf1 system[J]. Mol Plant, 2017, 10(7):1 011-1 013.
[21] HIEI Y, OHTA S, KOMARI T, et al. Efficient transformation of rice(Oryza sativa L.)mediated by agrobacterium and sequence analysis of the boundaries of the T-DNA[J]. Plant J,1994, 6(2):271–282.
[22] ZHANG G H, GAO M G, ZHANG G Z, et al. A high through-put protocol of plant genomic DNA Preparation for PCR[J]. Acta Agronomica Sinica, 2013, 39(7):1 200.
[23] XUE H Y, JI L J, GAO A M, et al. CRISPR-Cas9 for medical genetic screens:applications and future perspectives[J]. J Med Genet, 2016,53(2):91.
[24]李娟,常闪闪,刘凤权,等. Os DUF500基因沉默提高水稻对白叶枯病的抗性[J].中国水稻科学,2012,26(4):476-480.
[25]黄建平.水稻逆境诱导型启动子和低温应答基因Os LCL3的分离与鉴定[D].武汉:华中农业大学,2015.