棉花GhPAO3基因的CRISPR/Cas9表达载体的构建
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摘要
【目的】研究棉花GhPAO3基因的功能,通过CRISPR/Cas9技术构建棉花GhPAO3基因的基因编辑载体。【方法】筛选合适的两个PAM位点设计引物,在引物中添加接头,重叠延伸PCR,将两个靶标片段连接在一起。通过限制性核酸内切酶BsaⅠ对pRGEB32-7载体进行单酶切,使用一步法连接重叠延伸产物与线性化的pRGEB32-7载体。【结果】使用电击转化法将构建好的棉花GhPAO3基因的基因编辑载体转入农杆菌LBA4404感受态,通过卡那霉素筛选阳性单克隆菌株。【结论】条带大小与预期一致,棉花GhPAO3基因的基因编辑载体构建成功。
【Objective】 In order to further study the function of the cotton GhPAO3 gene, the gene editing vector for the cotton GhPAO3 gene was constructed by CRISPR/Cas9 technique.【Method】Primers were designed by screening the appropriate two PAM sites, and a linker was added to the primers, and the two target fragments were ligated together by overlap extension PCR. The pRGEB32-7 vector was subjected to single enzyme digestion by restriction endonuclease Bsa I, and the overlap extension product and the linearized pRGEB32-7 vector were ligated using a one-step method.【Results】The gene editing vector of the constructed cotton GhPAO3 gene was transferred into Agrobacterium LBA4404 competent state by electroporation transformation method and the positive monoclonal strain was screened by kanamycin.【Conclusion】The result of PCR showed that the band size was consistent with expectation, which indicated that the gene editing vector of cotton GhPAO3 gene had been constructed successfully.
【Objective】 In order to further study the function of the cotton GhPAO3 gene, the gene editing vector for the cotton GhPAO3 gene was constructed by CRISPR/Cas9 technique.【Method】Primers were designed by screening the appropriate two PAM sites, and a linker was added to the primers, and the two target fragments were ligated together by overlap extension PCR. The pRGEB32-7 vector was subjected to single enzyme digestion by restriction endonuclease Bsa I, and the overlap extension product and the linearized pRGEB32-7 vector were ligated using a one-step method.【Results】The gene editing vector of the constructed cotton GhPAO3 gene was transferred into Agrobacterium LBA4404 competent state by electroporation transformation method and the positive monoclonal strain was screened by kanamycin.【Conclusion】The result of PCR showed that the band size was consistent with expectation, which indicated that the gene editing vector of cotton GhPAO3 gene had been constructed successfully.
引文
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[8] 刘迪,刘琳琳,杜海英,等.CRISPR/Cas9植物基因编辑系统敲除棉花GhSBP基因表达载体的构建[J].分子植物育种,2018,16(1):135-139.LIU Di,LIU Lin-lin,DU Hai-ying,et al.(2018).Construction of Expression Vector of Knocking out GhSBP Gene in Cottonby CRISPR/Cas9 System [J].Molecular Plant Breeding,16(1):135-139.(in Chinese)
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[15] Baniasadi,F.,Saffari,V.R.,Moud,A.A.M.(2018).Physiological and growth responses of Calendula officinalis,L.plants to the interaction effects of polyamines and salt stress.Scientia Horticulturae,234:312-317.
[16] Yan,Z.,Zhou,L.,Ya-Ping,L.,Xin-Quan,Z.,Xiao,M.,& Lin-Kai,H.,et al.(2018).Chitosan and spermine enhance drought resistance in white clover,associated with changes in endogenous phytohormones and polyamines,and antioxidant metabolism.Functional Plant Biology.
[17] 李丽杰,顾万荣,孟瑶,等.干旱胁迫下亚精胺对玉米幼苗抗旱性影响的生理生化机制[J].应用生态学报,2018,29(2):554-564.LI Li-jie,GU Wan-rong,MENG Yao ,et al.(2018).Physiological and biochemical mechanism of spermidine improving drought resistance inmaize seedlings under drought stress [J].Chinese Journal of Applied Ecology,29(2):554-564.(in Chinese)
[18] Cheng,X.Q.,Zhu,X.F.,Tian,W.G.,Cheng,W.H.,Hakim,,& Sun,J.,et al.(2017).Genome-wide identification and expression analysis of polyamine oxidase genes in upland cotton (gossypium hirsutuml.).Plant Cell,Tissue and Organ Culture (PCTOC),129(2):237-249.
[19] 蒲艳,刘超,林琪,等.基于PCR方法对载体pBluescriptⅡSK(+)及pCAMBⅠA1300的定点突变[J].华北农学报,2016,31(6):83-87.PU Yan,LIU Chao,LIN Qi,et al.(2016).Site-directed Mutagenesis of pBluescript Ⅱ SK(+) and pCAMBⅠA1300 Vectors Based on PCR Method [J].Acta Agriculturae Boreali-Sinica,31(6):83-87.(in Chinese)
[20] 王尚堃,孙玲凌.多胺及其合成抑制剂对旱胁迫下杏幼苗叶片光合作用和游离态多胺含量的影响[J].江苏农业科学,2017,45(4):88-91.Wang Shang-kun,SUN Ling-ling.(2017).Effects of polyamine and its synthesis inhibitors on leaf photosynthesis and free polyamine content of apricot seedling under drought stress [J].Jiangsu Agricultural Sciences,45(4):88-91.(in Chinese)
[21] Voytas,& Daniel,F..(2013).Plant genome engineering with sequence-specific nucleases.Annual Review of Plant Biology,64(1):327-350.
[22] Chamberlain,& J.,R..(2004).Gene targeting in stem cells from individuals with osteogenesis imperfecta.Science,303(5661):1,198-1,201.
[23] Johnson,L.,Mercer,K.,Greenbaum,D.,Bronson,R.T.,Crowley,D.,& Tuveson,D.A.,et al.(2001).Somatic activation of the k-ras oncogene causes early onset lung cancer in mice.Nature,410(6832):1,111-1,116.
[2] 卢秀茹,贾肖月,牛佳慧.中国棉花产业发展现状及展望[J].中国农业科学,2018,51(1):26-36.LU Xiu-ru,JIA Xiao-yue,NIU Jia-hui.(2018).The Present Situation and Prospects of Cotton Industry Development in China[J].Scientia Agricultura Sinica,51(1):26-36.(in Chinese)
[3] 许艳超,韦洋洋,李振庆,等.复合盐碱胁迫下半野生棉苗期耐盐性综合评价及其关键生理指标的变化[J].棉花学报,2018,30(3):231-241.XU Yan-chao,WEI Yang-yang,LI Zhen-qing,et al.(2018).Integrated Evaluation and the Physiological and Biochemical Responses of Semi-Wild Cotton under Complex Salt-Alkali Stress [J].Cotton Science,30(3):231-241.(in Chinese)
[4] 王海标,陈全家,刘鹏鹏,等.苗期干旱胁迫对棉花生理特性、产量构成因素和纤维品质的影响[J].新疆农业科学,2013,50(12):2 172-2 181.WANG Hai-biao,CHEN Quan-jia,LIU Peng-peng ,et al.(2013).Effect of Drought during Seedling Stage on Physiological Traits,Yield Components and Fiber Quality of 4 Kinds of Cotton [J].Xinjiang Agricultural Sciences,50(12):2,172-2,181.(in Chinese)
[5] 徐立华.我国棉花高产、高效栽培技术研究现状与发展思路[J].中国棉花,2001,28(3):5-8.XU Li-hua.(2001).Research Status and Development Ideas of High Yield and High Efficient Cultivation Technology of Cotton in China [J].China Cotton,28(3):5-8.(in Chinese)
[6] Cao,H.X.,Wenqin,W.,Le,H.T.T.,& Vu,G.T.H.(2016).The power of crispr-cas9-induced genome editing to speed up plant breeding.International Journal of Genomics,2016,1-10.DOI:10.1155/2016/5078796
[7] Esvelt,K.M.,& Wang,H.H..(2013).Genome-scale engineering for systems and synthetic biology.mole syst biol 9:641.Molecular Systems Biology,9(1):641.
[8] 刘迪,刘琳琳,杜海英,等.CRISPR/Cas9植物基因编辑系统敲除棉花GhSBP基因表达载体的构建[J].分子植物育种,2018,16(1):135-139.LIU Di,LIU Lin-lin,DU Hai-ying,et al.(2018).Construction of Expression Vector of Knocking out GhSBP Gene in Cottonby CRISPR/Cas9 System [J].Molecular Plant Breeding,16(1):135-139.(in Chinese)
[9] Wiedenheft,B.,Sternberg,S.H.,& Doudna,J.A..(2012).Rna-guided genetic silencing systems in bacteria and archaea.NATURE,482(7385):331-338.
[10] Pennisi,E.(2013).The crispr craze.Science,341(6148):833-836.
[11] Li,C.,Unver,T.,& Zhang,B..(2017).A high-efficiency crispr/cas9 system for targeted mutagenesis in cotton (gossypium hirsutum l.).Scientific Reports,7,43902.
[12] Chen,X.,Lu,X.,Shu,N.,Wang,S.,Wang,J.,& Wang,D.,et al.(2017).Targeted mutagenesis in cotton (gossypium hirsutum l.) using the crispr/cas9 system.Scientific Reports,7,44304.
[13] Wang,P.,Zhang,J.,Sun,L.,Ma,Y.,& Zhang,X..(2017).High efficient multi-sites genome editing in allotetraploid cotton (gossypium hirsutum) using crispr/cas9 system.Plant Biotechnology Journal,16(1).
[14] Fincato,P.,Moschou,P.N.,Ahou,A.,Angelini,R.,Roubelakis-Angelakis,K.A.,& Federico,R.,et al.(2012).The members ofarabidopsis thalianapaogene family exhibit distinct tissue- and organ-specific expression pattern during seedling growth and flower development.Amino Acids ,42(2-3):831-841.
[15] Baniasadi,F.,Saffari,V.R.,Moud,A.A.M.(2018).Physiological and growth responses of Calendula officinalis,L.plants to the interaction effects of polyamines and salt stress.Scientia Horticulturae,234:312-317.
[16] Yan,Z.,Zhou,L.,Ya-Ping,L.,Xin-Quan,Z.,Xiao,M.,& Lin-Kai,H.,et al.(2018).Chitosan and spermine enhance drought resistance in white clover,associated with changes in endogenous phytohormones and polyamines,and antioxidant metabolism.Functional Plant Biology.
[17] 李丽杰,顾万荣,孟瑶,等.干旱胁迫下亚精胺对玉米幼苗抗旱性影响的生理生化机制[J].应用生态学报,2018,29(2):554-564.LI Li-jie,GU Wan-rong,MENG Yao ,et al.(2018).Physiological and biochemical mechanism of spermidine improving drought resistance inmaize seedlings under drought stress [J].Chinese Journal of Applied Ecology,29(2):554-564.(in Chinese)
[18] Cheng,X.Q.,Zhu,X.F.,Tian,W.G.,Cheng,W.H.,Hakim,,& Sun,J.,et al.(2017).Genome-wide identification and expression analysis of polyamine oxidase genes in upland cotton (gossypium hirsutuml.).Plant Cell,Tissue and Organ Culture (PCTOC),129(2):237-249.
[19] 蒲艳,刘超,林琪,等.基于PCR方法对载体pBluescriptⅡSK(+)及pCAMBⅠA1300的定点突变[J].华北农学报,2016,31(6):83-87.PU Yan,LIU Chao,LIN Qi,et al.(2016).Site-directed Mutagenesis of pBluescript Ⅱ SK(+) and pCAMBⅠA1300 Vectors Based on PCR Method [J].Acta Agriculturae Boreali-Sinica,31(6):83-87.(in Chinese)
[20] 王尚堃,孙玲凌.多胺及其合成抑制剂对旱胁迫下杏幼苗叶片光合作用和游离态多胺含量的影响[J].江苏农业科学,2017,45(4):88-91.Wang Shang-kun,SUN Ling-ling.(2017).Effects of polyamine and its synthesis inhibitors on leaf photosynthesis and free polyamine content of apricot seedling under drought stress [J].Jiangsu Agricultural Sciences,45(4):88-91.(in Chinese)
[21] Voytas,& Daniel,F..(2013).Plant genome engineering with sequence-specific nucleases.Annual Review of Plant Biology,64(1):327-350.
[22] Chamberlain,& J.,R..(2004).Gene targeting in stem cells from individuals with osteogenesis imperfecta.Science,303(5661):1,198-1,201.
[23] Johnson,L.,Mercer,K.,Greenbaum,D.,Bronson,R.T.,Crowley,D.,& Tuveson,D.A.,et al.(2001).Somatic activation of the k-ras oncogene causes early onset lung cancer in mice.Nature,410(6832):1,111-1,116.
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