紫花苜蓿MsSAG113基因的克隆及对拟南芥的转化
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摘要
以紫花苜蓿为试材,采用RT-PCR技术克隆出一个与拟南芥SAG113同源的基因,利用无缝连接酶将其与3302Y连接构建过量表达的植物表达载体,通过花序浸泡法转化拟南芥获得具有草铵膦抗性的植株,以期为研究MsSAG113的功能提供参考依据。结果表明:MsSAG113基因编码区长849bp,编码283个氨基酸,PCR和RT-PCR检测显示成功获得了转MsSAG113基因拟南芥植株,初步证明在转基因拟南芥中外源SAG113基因能够转录表达。
Alfalfa was used as material to clone a homologous gene named SAG113 in Arabidopsis thaliana by RT-PCR technique.The MsSAG113 gene was inserted into plasmid 3302 Y with seamless ligase constructing a plant expressing vector,and transgenic Arabidopsis thaliana plants with glufosinate-ammonium resistance were screened by floral dip method.The transgenic materials provide the foundation for further study of MsSAG113 gene.The results showed that its coding domain sequence was 849 bp in length encoding 283 amino acids,and PCR and RT-PCR analysis confirmed the transgenic Arabidopsis thaliana plants with MsSAG113 gene were successfully obtained,which preliminary proved transcription and expression of exogenous gene SAG113 in transgenic Arabidopsis thaliana was able.
Alfalfa was used as material to clone a homologous gene named SAG113 in Arabidopsis thaliana by RT-PCR technique.The MsSAG113 gene was inserted into plasmid 3302 Y with seamless ligase constructing a plant expressing vector,and transgenic Arabidopsis thaliana plants with glufosinate-ammonium resistance were screened by floral dip method.The transgenic materials provide the foundation for further study of MsSAG113 gene.The results showed that its coding domain sequence was 849 bp in length encoding 283 amino acids,and PCR and RT-PCR analysis confirmed the transgenic Arabidopsis thaliana plants with MsSAG113 gene were successfully obtained,which preliminary proved transcription and expression of exogenous gene SAG113 in transgenic Arabidopsis thaliana was able.
引文
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[25]王丹,王俊杰,赵彦.黄花苜蓿黄酮醇合成酶基因(MfFLS1)的克隆及生物信息学分析[J].中国草地学报,2016(1):20-26,53.
[26]王梦颖,晁跃辉,丛丽丽,等.紫花苜蓿MsARGOS基因的克隆及对拟南芥的转化[J].中国草地学报,2014(4):52-59.
[2]王建勇,姚晓华,张志斌.植物叶片衰老机理与调控研究进展[J].安徽农业科学,2011,39(31):19036-19038,19058.
[3]BREEZE E,HARRISON E,MCHATTIE S,et al.Highresolution temporal profiling of transcripts during Arabidopsis leaf senescence reveals a distinct chronology of processes and regulation[J].The Plant Cell,2011,23(3):873-894.
[4]JING H C,STURRE M J,HILLE J,et al.Arabidopsis onset of leaf death mutants identify a regulatory pathway controlling leaf senescence[J].Plant Journal,2002,32(1):51-63.
[5]LIM P O,KIM H J,NAM H G.Leaf senescence[J].Annual Review of Plant Biology,2007,58:115-136.
[6]WINGLER A,ROITSCH T.Metabolic regulation of leaf senescence:Interactions of sugar signaling with biotic and abiotic stress responses[J].Plant Biology,2008(10):50-62.
[7]GUO Y F,GAN S S,Convergence and divergence in gene expression profiles induced by leaf senescence and 27senescencepromoting hormonal,pathological and environmental stress treatments[J].Plant Cell Environ,2012,35:644-655.
[8]张兰,滕珂,尹淑霞.草地早熟禾叶绿素酶1基因PpCHL1的克隆和表达分析[J].中国草地学报,2016(4):1-7.
[9]CHEN H J,LIN Z W,HUANG G J,et al.Sweet potato calmodulin SPCAMis involved in salt stress-mediated leaf senescence,H2O2elevation and senescence-associated gene expression[J].Journal of Plant Physiology,2012,169(18):1892-1902.
[10]杨同文,李成伟.植物叶片衰老的表观遗传调控[J].植物学报,2014(6):729-737.
[11]周峰,华春,王仁雷.植物叶片衰老及调控[J].北方园艺,2012(1):171-172.
[12]GAN S S.Mitotic and postmitotic senescence in plants[J].Science of Aging Knowledge Environment,2003(38):E7.
[13]刘丹,唐加红,杨玉兰,等.干旱胁迫对银杏衰老期叶片生理生化特性的影响[J].北方园艺,2011(9):70-73.
[14]李静,薛鑫,吴金霞.转基因苜蓿的研究进展[J].生物技术通报,2012(11):1-8.
[15]刘晶晶,刘小平,师建芳,等.高温分解与乳酸菌分步发酵提高秸秆饲料消化率及适口性[J].农业工程学报,2014(22):290-299.
[16]ZHANG K,GAN S.An abscisic acid-AtNAP transcription factor-SAG113protein phosphatase 2Cregulatory chain for controlling dehydration in senescing Arabidopsis leaves[J].Plant Physiology,2012,158(4):961-969.
[17]范凯,王学德,袁淑娜,等.植物转录因子NAP亚家族的研究进展[J].中国农业科学,2014(2):209-220.
[18]ZHANG K W,XIA X Y,ZHANG Y Y,et al.An ABA-regulated and Golgi-localized protein phosphatase controls water loss during leaf senescence in Arabidopsis[J].The Plant Journal,2012,69(4):667-678.
[19]MORITA R,SATO Y,MASUDA Y,et al.Defect in nonyellow coloring 3,anα/βhydrolase-fold family protein,causes a stay-green phenotype during leaf senescence in rice[J].The Plant Journal,2009,59(6):940-952.
[20]敖特根白音,杨学举,郎明林,等.蒙古冰草半胱氨酸蛋白酶基因MwCP1的克隆[J].中国草地学报,2015(5):11-17.
[21]晁跃辉,李珊珊,滕珂,等.紫花苜蓿MsNAP基因克隆及烟草转化[J].中国草地学报,2016(2):13-19.
[22]ALEXANDROV N N,BROVER V V,FREIDIN S,et al.Insights into corn genes derived from large-scale cDNA sequencing[J].Plant Molecular Biology,2009,69(1-2):179-194.
[23]PARK S Y,YU J W,PARK J S,et al.The senescenceinduced staygreen protein regulates chlorophyll degradation[J].Plant Cell,2007,19(5):1649-1664.
[24]REN G D,AN K,LIAO Y,et al.Identification of a novel chloroplast protein AtNYE1regulating chlorophyll degradation during leaf senescence in Arabidopsis[J].Plant Physiology,2007,144(3):1429-1441.
[25]王丹,王俊杰,赵彦.黄花苜蓿黄酮醇合成酶基因(MfFLS1)的克隆及生物信息学分析[J].中国草地学报,2016(1):20-26,53.
[26]王梦颖,晁跃辉,丛丽丽,等.紫花苜蓿MsARGOS基因的克隆及对拟南芥的转化[J].中国草地学报,2014(4):52-59.