水稻膜联蛋白基因OsAnn8干旱和低温条件下表达模式以及CRISPR/Cas9定点编辑
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摘要
为了阐明水稻膜联蛋白基因家族成员OsAnn8在干旱和低温胁迫条件下的功能作用机制,通过荧光定量PCR技术对不同干旱和低温处理下的水稻叶片中OsAnn8基因进行转录水平上的定量分析,结果表明,OsAnn8基因在干旱胁迫处理前后表达量呈现出高-低-高的变化趋势,而在冷胁迫处理前后表达量则呈现出低-高-低-低的变化。随后,利用CRISPR/Cas9基因编辑技术对OsAnn8基因进行定点编辑,并借助农杆菌介导将OsAnn8基因靶位点的CRISPR/Cas9基因编辑植物表达载体导入转基因受体品种TP309,经潮霉素筛选后共获得32株转基因阳性植株,靶位点扩增测序峰图分析表明其中的2个单株出现了重叠峰,进一步的T载体克隆分别测序表明,这2个单株为单等位基因敲除,说明本研究已经成功获得了OsAnn8基因靶位点敲除的单等位突变体,不同类型的水稻OsAnn8基因突变体的创建,为水稻膜联蛋白基因家族成员OsAnn8在非生物胁迫条件下的功能研究奠定了材料基础。
In order to clarify the functional mechanism of rice annexin OsAnn8 under drought and low temperature stress,the transcription level of OsAnn8 in rice leaves was analysed by fluorescence quantitative PCR technology. The results showed that the expression of OsAnn8 gene presented a high-low-high trend before and after drought stress,but a low-high-low-low changes before and after cold stress. Subsequently,the knocking out of OsAnn8 was also performed using the CRISPR/Cas9 gene editing techniques,and the CRISPR/Cas9 gene editing plant expression vector was transformed into the transgenic receptor variety TP309 by the agrobacterium-mediated method. A total of 32 transgenic positive plants were obtained by selection for hygromycin resistance. The analysis of sequencing map in target locus showed that overlapping peaks existed in two individual plants. Furthermore,T vector clones were sequenced separately,indicating that the monoallelic mutation happened in the two individual plants. The results indicated that monoallelic mutants had been obtained at OsAnn8 gene locus successfully. The creation of different types of mutants at OsAnn8 gene locus of rice laid the material base for gene function research under abiotic stress.
In order to clarify the functional mechanism of rice annexin OsAnn8 under drought and low temperature stress,the transcription level of OsAnn8 in rice leaves was analysed by fluorescence quantitative PCR technology. The results showed that the expression of OsAnn8 gene presented a high-low-high trend before and after drought stress,but a low-high-low-low changes before and after cold stress. Subsequently,the knocking out of OsAnn8 was also performed using the CRISPR/Cas9 gene editing techniques,and the CRISPR/Cas9 gene editing plant expression vector was transformed into the transgenic receptor variety TP309 by the agrobacterium-mediated method. A total of 32 transgenic positive plants were obtained by selection for hygromycin resistance. The analysis of sequencing map in target locus showed that overlapping peaks existed in two individual plants. Furthermore,T vector clones were sequenced separately,indicating that the monoallelic mutation happened in the two individual plants. The results indicated that monoallelic mutants had been obtained at OsAnn8 gene locus successfully. The creation of different types of mutants at OsAnn8 gene locus of rice laid the material base for gene function research under abiotic stress.
引文
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[8] Clark G B,Sessions A,Eastburn D J,Roux S J. Differ-ential expression of members of the annexin multigenefamily in Arabidopsis[J]. Plant Physiology,2001,126(3):1072-1084.
[9] Hofmann A. Annexins in the plant kingdom:Perspectivesand potentials[J]. Annexins,2004,1(1):51-61.
[10] Huh S M,Noh E K,Kim H G,Jeon B W,Bae K,HuH C,Kwak J M,Park O K. Arabidopsis annexins An-n At1 and Ann At4 interact with each other and regulatedrought and salt stress responses[J]. Plant and CellPhysiology,2010,51(9):1499-1514. doi:10. 1093/pcp/pcq111.
[11] Jia F J,Wang C Y,Huang J,Yang G D,Wu C A,Zheng C C. SCF E3 ligase PP2-B11 plays a positive rolein response to salt stress in Arabidopsis[J]. Journal ofExperimental Botany,2015,66(15):4683-4697. doi:10. 1093/jxb/erv245.
[12] Wang X,Ma X L,Wang H,Li B J,Clark G,Guo Y,Roux S,Sun D Y,Tang W Q. Proteomic study of micro-somal proteins reveals a key role for arabidopsis annexin1 in mediating heat stress-induced increase in intracellu-lar calcium levels[J]. Molecular&Cellular Proteomics,2015,14(3):686-694. doi:10. 1074/mcp. M114.042697.
[13] Zhu J E,Wu X R,Yuan S J,Qian D,Nan Q,An LZ,Xiang Y. Annexin5 plays a vital role in Arabidopsispollen development via Ca2+-Dependent membrane traf-ficking[J]. PLo S One,2014,9(7):e102407. doi:10.1371/journal. pone. 0102407.
[14] Zhu J E,Yuan S J,Wei G,Qian D,Wu X R,Jia HL,Gui M Y,Liu W Z,An L Z,Xiang Y. Annexin5 isessential for pollen development in Arabidopsis[J]. Mo-lecular Plant,2014,7(4):751-754. doi:10. 1093/mp/sst171.
[15] Mortimer J C,Laohavisit A,Macpherson N,Webb A,Brownlee C,Battey N H,Davies J M. Annexins:multi-functional components of growth and adaptation[J].Journal of Experimental Botany,2008,59(3):533-544. doi:10. 1093/jxb/erm344.
[16] Konopka-Postupolska D,Clark G,Goch G,Debski J,Floras K,Cantero A,Fijolek B,Roux S,Hennig J. Therole of annexin 1 in drought stress in Arabidopsis[J].Plant Physiology,2009,150(3):1394-1410. doi:10.1104/pp. 109. 135228.
[17] Lee S,Lee E J,Yang E J,Lee J E,Park A R,Song WH,Park O K. Proteomic identification of annexins,cal-cium-dependent membrane binding proteins that mediateosmotic stress and abscisic acid signal transduction inArabidopsis[J]. The Plant Cell,2004,16(6):1378-1391. doi:10. 1105/tpc. 021683.
[18] Qiao B,Zhang Q,Liu D L,Wang H Q,Yin J Y,Wang R,He M L,Cui M,Shang Z L,Wang D K,ZhuZ G. A calcium-binding protein,rice annexin Os ANN1,enhances heat stress tolerance by modulating the pro-duction of H2O2[J]. Journal of Experimental Botany,2015,66(19):5853-5866. doi:10. 1093/jxb/erv294.
[19] Zhou M L,Yang X B,Zhang Q,Zhou M,Zhao E Z,Tang Y X,Zhu X M,Shao J R,Wu Y M. Induction ofannexin by heavy metals and jasmonic acid in Zea mays[J]. Functional&Integrative Genomics,2013,13(2):241-251. doi:10. 1007/s10142-013-0316-5.
[20] Jami S K,Clark G B,Ayele B T,Roux S J,Kirti P B.Identification and characterization of annexin gene familyin rice[J]. Plant Cell Reports,2012,31(5):813-825.doi:10. 1007/s00299-011-1201-0.
[21] Shen C X,Que Z,Xia Y M,Tang N,Li D,He R H,Cao M L. Knock out of the annexin gene Os Ann3 viaCRISPR/Cas9-mediated genome editing decreased coldtolerance in rice[J]. Journal of Plant Biology,2017,60(6):539-547. doi:10. 1007/s12374-016-0400-1.
[22]沈春修.水稻LOC_Os10g05490位点冷胁迫条件下表达分析及CRISPR/Cas9定向编辑[J].浙江农业学报,2017,29(2):177-185.Shen C X. CRISPR/Cas9 editing and expression analy-sis of LOC_Os10g05490 locus under cold stress[J].Acta Agriculturae Zhejiangensis,2017,29(2):177-185.
[23]李丁.以潮霉素为筛选标记的水稻叶绿体转化体系的建立[D].长沙:中南大学,2013.Li D. Establishment of the chloroplst genetic transforma-tion system in rice by using hygromycin B as selectionpressure[D]. Changsha:Zhongnan University,2013.
[24] Creutz C E,Pazoles C J,Pollard H B. Identification andpurification of an adrenal medullary protein(synexin)that causes calcium-dependent aggregation of isolatedchromaffin granules[J]. The Journal of Biological Chem-istry,1978,253(8):2858-2866.
[25] Cantero A,Barthakur S,Bushart T J,Chou S,MorganR O,Fernandez M P,Clark G B,Roux S J. Expressionprofiling of the Arabidopsis annexin gene family duringgermination,de-etiolation and abiotic stress[J]. PlantPhysiology and Biochemistry,2006,44(1):13-24. doi:10. 1016/j. plaphy. 2006. 02. 002.
[26] Fowler S,Thomashow M F. Arabidopsis transcriptomeprofiling indicates that multiple regulatory pathways areactivated during cold acclimation in addition to the CBFcold response pathway[J]. The Plant Cell,2002,14(8):1675-1690. doi:10. 1105/tpc. 003483.
[2] Apel K,Hirt H. Reactive oxygen species:metabolism,oxidative stress,and signal transduction[J]. Annual Re-view of Plant Biology,2004,55:373-399. doi:10. 1146/annurev. arplant. 55. 031903. 141701.
[3] Clark G B,Morgan R O,Fernandez M P,Roux S J. Evo-lutionary adaptation of plant annexins has diversified theirmolecular structures, interactions and functional roles[J]. The New Phytologist,2012,196(3):695-712. doi:10. 1111/j. 1469-8137. 2012. 04308. x.
[4] Moss S E,Morgan R O. The annexins[J]. Genome Biolo-gy,2004,5(4):219. doi:10. 1186/gb-2004-5-4-219.
[5] Davies J. Annexin-mediated calcium signalling in plants[J]. Plants,2014,3(1):128-140.
[6] Gerke V,Creutz C E,Moss S E. Annexins:linking Ca2+signalling to membrane dynamics[J]. Nature ReviewsMolecular Cell Biology,2005,6(6):449-461. doi:10.1038/nrm1661.
[7] Jami S K,Clark G B,Ayele B T,Ashe P,Kirti P B. Ge-nome-wide comparative analysis of annexin superfamily inplants[J]. PLo S One,2012,7(11):e47801. doi:10.1371/journal. pone. 0047801.
[8] Clark G B,Sessions A,Eastburn D J,Roux S J. Differ-ential expression of members of the annexin multigenefamily in Arabidopsis[J]. Plant Physiology,2001,126(3):1072-1084.
[9] Hofmann A. Annexins in the plant kingdom:Perspectivesand potentials[J]. Annexins,2004,1(1):51-61.
[10] Huh S M,Noh E K,Kim H G,Jeon B W,Bae K,HuH C,Kwak J M,Park O K. Arabidopsis annexins An-n At1 and Ann At4 interact with each other and regulatedrought and salt stress responses[J]. Plant and CellPhysiology,2010,51(9):1499-1514. doi:10. 1093/pcp/pcq111.
[11] Jia F J,Wang C Y,Huang J,Yang G D,Wu C A,Zheng C C. SCF E3 ligase PP2-B11 plays a positive rolein response to salt stress in Arabidopsis[J]. Journal ofExperimental Botany,2015,66(15):4683-4697. doi:10. 1093/jxb/erv245.
[12] Wang X,Ma X L,Wang H,Li B J,Clark G,Guo Y,Roux S,Sun D Y,Tang W Q. Proteomic study of micro-somal proteins reveals a key role for arabidopsis annexin1 in mediating heat stress-induced increase in intracellu-lar calcium levels[J]. Molecular&Cellular Proteomics,2015,14(3):686-694. doi:10. 1074/mcp. M114.042697.
[13] Zhu J E,Wu X R,Yuan S J,Qian D,Nan Q,An LZ,Xiang Y. Annexin5 plays a vital role in Arabidopsispollen development via Ca2+-Dependent membrane traf-ficking[J]. PLo S One,2014,9(7):e102407. doi:10.1371/journal. pone. 0102407.
[14] Zhu J E,Yuan S J,Wei G,Qian D,Wu X R,Jia HL,Gui M Y,Liu W Z,An L Z,Xiang Y. Annexin5 isessential for pollen development in Arabidopsis[J]. Mo-lecular Plant,2014,7(4):751-754. doi:10. 1093/mp/sst171.
[15] Mortimer J C,Laohavisit A,Macpherson N,Webb A,Brownlee C,Battey N H,Davies J M. Annexins:multi-functional components of growth and adaptation[J].Journal of Experimental Botany,2008,59(3):533-544. doi:10. 1093/jxb/erm344.
[16] Konopka-Postupolska D,Clark G,Goch G,Debski J,Floras K,Cantero A,Fijolek B,Roux S,Hennig J. Therole of annexin 1 in drought stress in Arabidopsis[J].Plant Physiology,2009,150(3):1394-1410. doi:10.1104/pp. 109. 135228.
[17] Lee S,Lee E J,Yang E J,Lee J E,Park A R,Song WH,Park O K. Proteomic identification of annexins,cal-cium-dependent membrane binding proteins that mediateosmotic stress and abscisic acid signal transduction inArabidopsis[J]. The Plant Cell,2004,16(6):1378-1391. doi:10. 1105/tpc. 021683.
[18] Qiao B,Zhang Q,Liu D L,Wang H Q,Yin J Y,Wang R,He M L,Cui M,Shang Z L,Wang D K,ZhuZ G. A calcium-binding protein,rice annexin Os ANN1,enhances heat stress tolerance by modulating the pro-duction of H2O2[J]. Journal of Experimental Botany,2015,66(19):5853-5866. doi:10. 1093/jxb/erv294.
[19] Zhou M L,Yang X B,Zhang Q,Zhou M,Zhao E Z,Tang Y X,Zhu X M,Shao J R,Wu Y M. Induction ofannexin by heavy metals and jasmonic acid in Zea mays[J]. Functional&Integrative Genomics,2013,13(2):241-251. doi:10. 1007/s10142-013-0316-5.
[20] Jami S K,Clark G B,Ayele B T,Roux S J,Kirti P B.Identification and characterization of annexin gene familyin rice[J]. Plant Cell Reports,2012,31(5):813-825.doi:10. 1007/s00299-011-1201-0.
[21] Shen C X,Que Z,Xia Y M,Tang N,Li D,He R H,Cao M L. Knock out of the annexin gene Os Ann3 viaCRISPR/Cas9-mediated genome editing decreased coldtolerance in rice[J]. Journal of Plant Biology,2017,60(6):539-547. doi:10. 1007/s12374-016-0400-1.
[22]沈春修.水稻LOC_Os10g05490位点冷胁迫条件下表达分析及CRISPR/Cas9定向编辑[J].浙江农业学报,2017,29(2):177-185.Shen C X. CRISPR/Cas9 editing and expression analy-sis of LOC_Os10g05490 locus under cold stress[J].Acta Agriculturae Zhejiangensis,2017,29(2):177-185.
[23]李丁.以潮霉素为筛选标记的水稻叶绿体转化体系的建立[D].长沙:中南大学,2013.Li D. Establishment of the chloroplst genetic transforma-tion system in rice by using hygromycin B as selectionpressure[D]. Changsha:Zhongnan University,2013.
[24] Creutz C E,Pazoles C J,Pollard H B. Identification andpurification of an adrenal medullary protein(synexin)that causes calcium-dependent aggregation of isolatedchromaffin granules[J]. The Journal of Biological Chem-istry,1978,253(8):2858-2866.
[25] Cantero A,Barthakur S,Bushart T J,Chou S,MorganR O,Fernandez M P,Clark G B,Roux S J. Expressionprofiling of the Arabidopsis annexin gene family duringgermination,de-etiolation and abiotic stress[J]. PlantPhysiology and Biochemistry,2006,44(1):13-24. doi:10. 1016/j. plaphy. 2006. 02. 002.
[26] Fowler S,Thomashow M F. Arabidopsis transcriptomeprofiling indicates that multiple regulatory pathways areactivated during cold acclimation in addition to the CBFcold response pathway[J]. The Plant Cell,2002,14(8):1675-1690. doi:10. 1105/tpc. 003483.