TALEN介导水稻OsTZF9基因定点突变研究
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摘要
RR-TZF锌指基因在植物生长发育和胁迫反应中起着重要作用。利用TALEN基因组编辑技术定点突变水稻RR-TZF基因OsTZF9,结果表明:根据日本晴基因组中的OsTZF9基因序列,设计3对TALEN靶序列,并构建相应的3个TALEN植物敲除表达载体;通过农杆菌介导法分别将3个TALEN敲除载体导入水稻明恢86和秀水134的胚性愈伤组织中,获得了100个转化再生植株,PCR检测获得87个阳性植株;PCR扩增转基因植株靶突变区并测序列,发现所有转基因植株均未出现靶突变。
RR-TZF zinc finger gene plays an important role in plant growth, development and stress response. Target mutation of RR-TZF gene OsTZF9 in rice was carried out by TALEN genome editing technique. The results showed that three pairs of TALEN target sequences were designed according to the sequence of OsTZF9 gene in Nipponbare, and three plant knockout expression vectors were constructed and introduced into embryogenic callus of Minghui 86 and Xiushui 134 by Agrobacterium-mediated transformation, obtaining 100 transformants. Among them, 87 positive plants were obtained through PCR detection. The target mutation regions of the transgenic plants were detected and sequenced, to find no target mutation.
RR-TZF zinc finger gene plays an important role in plant growth, development and stress response. Target mutation of RR-TZF gene OsTZF9 in rice was carried out by TALEN genome editing technique. The results showed that three pairs of TALEN target sequences were designed according to the sequence of OsTZF9 gene in Nipponbare, and three plant knockout expression vectors were constructed and introduced into embryogenic callus of Minghui 86 and Xiushui 134 by Agrobacterium-mediated transformation, obtaining 100 transformants. Among them, 87 positive plants were obtained through PCR detection. The target mutation regions of the transgenic plants were detected and sequenced, to find no target mutation.
引文
[1]POMERANZ MC,HAH C,LIN PC,et al.The Arabidopsis tandem zinc finger protein AtTZF1 traffics between the nucleus and cytoplasmic foci and binds both DNA and RNA[J].Plant Physiol,2010,152:151-165.
[2]Wang D,Guo Y,Wu C,et al.Genome-wide analysis of CCCH zinc finger family in Arabidopsis and rice[J].BMC Genomics,2008,9:44-54.
[3]刘梅芳,周淑芬,徐桂磊,等.一个水稻CCCH型锌指蛋白基因的表达模式分析[J].福建农业学报,2008,23(3):225-230.
[4]周淑芬,刘华清,徐桂磊,等.水稻种子发育期间特异性锌指蛋白基因的筛选与分析[J].中国生物化学与分子生物学报,2012,9(2):237-289.
[5]CHEN Y,SUN A,WANG M,et al.Functions of the CCCH type zinc finger protein OsGZF1 in regulation of the seed storage protein GluB 1from rice[J].Plant Mol Biol,2014,84:621-634.
[6]POMERANZ M,FINER J,JANG JC.Putative molecular mechanisms underlying tandem CCCH zinc finger protein mediated plant growth,stress and gene expression responses[J].Plant Signaling & Behavior,2011,6(5):647-651.
[7]MILLER JC,TAN S,QIAO G,et al.A TALE nuclease architecture for efficient genome editing[J].Nat Biotechnol,2011,29:143-148.
[8]苏军,胡昌泉,翟红利,等.农杆菌介导籼稻明恢86高效稳定转化体系的建立[J].福建农业学报,2003,18(4):209-213.
[9]MURRY MG,THOMPSON WF.Rapid isolation of high molecular weight plant DNA[J].Nucl Acid Res,1980,8(19):4321-4326.
[2]Wang D,Guo Y,Wu C,et al.Genome-wide analysis of CCCH zinc finger family in Arabidopsis and rice[J].BMC Genomics,2008,9:44-54.
[3]刘梅芳,周淑芬,徐桂磊,等.一个水稻CCCH型锌指蛋白基因的表达模式分析[J].福建农业学报,2008,23(3):225-230.
[4]周淑芬,刘华清,徐桂磊,等.水稻种子发育期间特异性锌指蛋白基因的筛选与分析[J].中国生物化学与分子生物学报,2012,9(2):237-289.
[5]CHEN Y,SUN A,WANG M,et al.Functions of the CCCH type zinc finger protein OsGZF1 in regulation of the seed storage protein GluB 1from rice[J].Plant Mol Biol,2014,84:621-634.
[6]POMERANZ M,FINER J,JANG JC.Putative molecular mechanisms underlying tandem CCCH zinc finger protein mediated plant growth,stress and gene expression responses[J].Plant Signaling & Behavior,2011,6(5):647-651.
[7]MILLER JC,TAN S,QIAO G,et al.A TALE nuclease architecture for efficient genome editing[J].Nat Biotechnol,2011,29:143-148.
[8]苏军,胡昌泉,翟红利,等.农杆菌介导籼稻明恢86高效稳定转化体系的建立[J].福建农业学报,2003,18(4):209-213.
[9]MURRY MG,THOMPSON WF.Rapid isolation of high molecular weight plant DNA[J].Nucl Acid Res,1980,8(19):4321-4326.