拟南芥bZIP23基因过量表达载体的构建及过表达植株的筛选

2014-04-29 00:44顾菊等
安徽农业科学 2014年14期
关键词:拟南芥

顾菊等

摘要 [目的]以拟南芥为材料克隆bZIP23基因,构建bZIP23基因的过量表达载体和筛选过表达植株,为验证其功能奠定基础。[方法]提取拟南芥总RNA和RTPCR克隆bZIP23基因,用限制性内切酶切割和T4 DNA连接酶连接,使bZIP23基因连接到35S强启动子的pART27载体上;将连接产物转化到Trans1T1感受态细胞中,筛选阳性单克隆进行菌落PCR鉴定并测序验证,获得重组质粒。将该重组质粒电激转化至根瘤农杆菌GV3101菌株,浸花法转化拟南芥野生型植株。[结果]通过单菌落PCR鉴定和DNA测序结果显示,bZIP23基因与35S过量表达载体已连接,获得了重组载体;抗性筛选与遗传鉴定获得相应的转基因过量表达阳性植株。[结论]构建的过量表达载体及筛选得到的过量表达植株为验证bZIP23基因功能奠定了基础。

关键词拟南芥(Arabidopsis athaliana);bZIP23;互补和过量表达载体;转基因植株

中图分类号Q754;S188文献标识码A文章编号0517-6611(2014)14-04199-03

Construction of Arabidopsis Gene bZIP23 Overexpression Vector and Screening of Expression Plant

GU Ju,CAO Shuqing et al(School of Biotechnology and Food Engineering,Hefei University of Technology,Hefei,Anhui 230009)

Abstract[Objective] Arabidopsis were used as material to clone bZIP23 gene,we construct gene bZIP23 overexpression vector and screen expression plant.[Method] Total RNA was extracted from Arabidopsis seedlings,and cDNA fragments of bZIP23 gene were amplified by RTPCR.Using the restriction enzymes and T4 DNA ligase,cDNA fragments were subsequently cloned into PART27 vectors,and then were transformed into TransT1 phage resistant chemically competent cells.Analysis of bacterial colony PCR and cDNA sequencing were performed to confirm that cDNA of the Arabidopsis thaliana bZIP23 gene was successfully cloned.The recombinant plasmids were obtained and transformed into Agrobacterium GV3101 cells.Wildtype Arabidopsis thaliana was transformed by using floraldip method.[Result] Analysis of bacterial colony PCR and DNA sequencing were performed to confirm recombinant plasmids,complementary and overexpression positive plants were obtained through genetic screening and identification of genetically modified methods.[Conclusion] Construction of Arabidopsis gene bZIP23 overexpression vector and screening of expression plant laid the foundation for the function of gene bZIP23.

Key wordsArabidopsis thaliana;bZIP23; Complementary and overexpression vector; Genetically modified plant

转录因子是一类识别DNA特异序列且结合在目的基因启动子的特定位点并调节其转录活性的蛋白,在拟南芥中鉴定了1 600多个转录因子[1]。有文献报道,转录因子bZIP23对重金属锌缺乏的响应[2]。转录因子对生物胁迫和非生物的响应,调节拟南芥对重金属的耐受[3]分子机理有重要作用[4]。bZIP家族有很多成员[5]。bZIP23是水稻碱性亮氨酸拉链[6](basic leucine zipper,bZIP)转录因子家族的成员之一,是ABA依赖的抗旱耐盐反应中主要调节因子,在通过遗传改良提高作物抗逆性中具有潜在的应用价值。cDNA全长1 828 bp,包含4个外显子,编码一个由357氨基酸组成的蛋白产物,产物含有碱性亮氨酸拉链(bZIP)結构域。bZIP23突变体:TDNA插入第2个内含子。过量表达[7]bZIP23的转基因水稻植株的抗旱性和耐盐性显著提高,同时增加ABA的敏感性[8];bZIP23突变体对ABA 的敏感性降低,抗旱性和耐盐性也显著降低。笔者构建基因35S:bZIP23过量表达载体,以期为进一步研究转录因子[9]调节拟南芥对重金属[10]耐受的分子机理提供依据。

1材料与方法

1.1材料

1.1.1研究对象。哥伦比亚野生型拟南芥(Arabidopsis athaliana)(Col0),购自美国拟南芥种质资源中心,由实验室繁衍保存。

1.1.2主要试剂。RevertAid First Strand cDNA Synthesis Kit、RNAiso Plus和PrimeSTAR MAX DNA Polymerase,均购自takara公司;T4 DNA连接酶、Easy Taq、TIANGel MiDi Purification Kit、TIANquick MiDi Purification Kit,TIANprep MiniPlasmid kit、XhoI和EcoRI,均购自NEB公司。

1.1.3宿主菌和载体。平末端载体pEASYblunt Simple Cloning Kit、感受态细胞Trans1T1 Chemically Competent Cell和pART27,由实验室繁衍保存。

1.2方法

图5获得过表达植株PCR电泳结果3结论与讨论

基因差异表达谱数据库中显示bZIP23基因表达水平受到铅胁迫诱导,表明该基因可能参与铅胁迫响应。为进一步研究该基因功能,试验从美国种子资源库获得了该基因敲除的突变体bZIP231和bZIP232,并利用分子生物学方法获得了bZIP23基因的过量表达植株。过量表达植株的获得将有利于研究植物对重金属的响应,在今后的研究中可能利用转基因植株解决重金属污染问题。随着一些抗逆基因的鉴定和抗逆机理不断深入研究,利用转基因技术将外源抗逆基因导入植物基因组,该技术在提高植物抗逆性、改作物遗传性状及培育农作物优良品系等方面具有广阔的应用前景[12-13]。

参考文献

[1] 刘强,张贵友.植物转录因子的结构与调控作用[J].科学通报,2000,45(14):1465-1474.

[2] ASSUNCA~OA A G L,HERREROA E.Arabidopsis thaliana transcription factors bZIP19 and bZIP23 regulate the adaptation to zinc deficiency [J].PNAS,2010,22:10296-10301.

[3] VANNINI C,LOCATELLI F,BRACALE M,et al.Overexpession of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants[J].Plant Journal,2001,37:115-127.

[4] CHEN Y H,YANG X Y,HE K,et al.The MYB transcription factor superfamily of Arabidopsis:expression analysis and phylogenetic comparison with the rice MYB family[J].Plant Molecular Biology,2006,60:107-124.

[5] JAKOBY M,WEISSHAAR B,DRGELASER W.bZIP transcription factors in Arabidopsis[J].Trends Plant Sci,2002,7(3):106- 111.

[6] RIECHMANN J L,HEARD J,MARTIN G,et al.Arabidopsis transcription factors:genomewide comparative analysis among eukaryotes [J].Science,2000,290(5499):2105-2110.

[7] NIJHAWAN A,JAIN M,AKHILESH K,et al.Genomic Survey and gene expression analysis of the basic Leucine Zipper transcription factor family in Rice [J].Plant Physiology2008,146(2):333-350.

[8] YONG X,NING T,HAO D,et al.Characterization of OsbZIP23 as a key player of the basic leucine zipper transcription factor family for conferring abscisic acid sensitivity and salinity and drought tolerance in rice [J].Plant Physiology,2008,148(4):1938-1952.

[9] PABO C O,SAUER R T.Transcription factors:structural families and principles of DNA recognition[J].Annual Review of Biochemistry,1992,61(10):1053-1095.

[10] CLEMENS S.Molecular mechanisms of plant metal tolerance and homeostasis[J].Planta,2011,212(4):475-486.

[11] 柏晓婭,顾菊,陈光朗,等.拟南芥抗旱突变体csm11的鉴定及其基因克隆[J].合肥工业大学学报,2013,36(12):1518-1522.

[12] 侯文胜,郭三堆,路明,等.利用转基因技术进行植物遗传改良[J].生物技术通报,2002(1):10-15.

[13] 吴福彪.基因工程与植物的遗传改良[J].生物学通报,2010,45(5):7-10.

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