Cloning and expression profiles of 15 genes encoding WRKY transcription factor in wheat （Triticum aestivem L.）

WRKY proteins are involved in various physiological processes, including biotic and abiotic stress responses, hormone responses and development. However, no systematic identification, expression and function analysis of WRKY genes in wheat were reported. In this study, we isolated 15 wheat cDNAs with complete open reading frame （ORF） encoding putative WRKY proteins using in silico cloning. Phylogenetic analysis indicated that the 15 wheat WRKY genes belonged to three major WRKY groups. Expression analysis revealed that most genes expressed drastically in leaf, except Ta WRKYIO which expressed in crown intensively. Four genes were strongly up-regulated with the senescence of leaves. Eight genes were responsive to low temperature, high temperature, NaCl or PEG treatment. Moreover, differential expression patterns were also observed between wheat hybrid and its parents, and some genes were more responsive to PEG treatment in the hybrid. These results demonstrated that wheat WRKY genes are involved in leaf senescing and abiotic stresses. And the changed expression of these WRKY genes in hybrid might contribute to the heterosis by improving the stress tolerance in hybrids.     

Cloning and expression pro?les of 15 genes encoding WRKY transcription factor in wheat (Triticum aestivem L.)

WRKY proteins are involved in various physiological processes, including biotic and abiotic stress responses, hormone responses and development. However, no systematic identi?cation, expression and function analysis of WRKY genes in wheat were reported. In this study, we isolated 15 wheat cDNAs with complete open reading frame (ORF) encoding putative WRKY proteins using in silico cloning. Phylogenetic analysis indicated that the 15 wheat WRKY genes belonged to three major WRKY groups. Expression analysis revealed that most genes expressed drastically in leaf, except TaWRKY10 which expressed in crown intensively. Four genes were strongly up-regulated with the senescence of leaves. Eight genes were responsive to low temperature, high temperature, NaCl or PEG treatment. Moreover, differential expression patterns were also observed between wheat hybrid and its parents, and some genes were more responsive to PEG treatment in the hybrid. These results demonstrated that wheat WRKY genes are involved in leaf senescing and abiotic stresses. And the changed expression of these WRKY genes in hybrid might contribute to the heterosis by improving the stress tolerance in hybrids. 2007 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.     

Cloning and expression pro?les of 15 genes encoding WRKY transcription factor in wheat (Triticum aestivem L.)

WRKY proteins are involved in various physiological processes, including biotic and abiotic stress responses, hormone responses and development. However, no systematic identi?cation, expression and function analysis of WRKY genes in wheat were reported. In this study, we isolated 15 wheat cDNAs with complete open reading frame (ORF) encoding putative WRKY proteins using in silico cloning. Phylogenetic analysis indicated that the 15 wheat WRKY genes belonged to three major WRKY groups. Expression analysis revealed that most genes expressed drastically in leaf, except TaWRKY10 which expressed in crown intensively. Four genes were strongly up-regulated with the senescence of leaves. Eight genes were responsive to low temperature, high temperature, NaCl or PEG treatment. Moreover, differential expression patterns were also observed between wheat hybrid and its parents, and some genes were more responsive to PEG treatment in the hybrid. These results demonstrated that wheat WRKY genes are involved in leaf senescing and abiotic stresses. And the changed expression of these WRKY genes in hybrid might contribute to the heterosis by improving the stress tolerance in hybrids. 2007 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.     

水稻转录因子WRKY39的克隆表达及DNA结合活性分析

根据水稻转录因子WRKY39的cDNA基因序列设计引物,克隆水稻日本晴(Oryza sativa L.)WRKY类转录因子的cDNA基因并进行原核表达,通过凝胶阻滞实验分析融合蛋白与DNA的结合活性。结果克隆了1个新的WRKY基因OsWRKY39,其编码的蛋白OsWRKY39能够与顺式元件W盒特异结合,Os-WRKY39具有转录因子的最基本特征。     

伴矿景天WRKY基因家族鉴定及镉胁迫响应分析

【目的】WRKY转录因子在植物响应非生物胁迫过程中发挥着重要的调控作用,目前超积累植物伴矿景天(Sedum plumbizincicola) WRKY转录因子的研究较少。进行SpWRKY基因家族的全基因组鉴定及其在镉胁迫下的表达模式分析可为后续SpWRKYs基因克隆和耐镉功能分析提供参考。【方法】对伴矿景天WRKY基因家族进行全基因组鉴定和生物信息学分析,利用转录组数据和qRT-PCR技术检测WRKY基因在镉胁迫下的表达模式并进行转基因拟南芥耐镉性的分析。【结果】伴矿景天基因组中共鉴定出77个SpWRKYs,非均匀地分布在各条染色体上;系统进化树分析发现,伴矿景天WRKY成员被分成3大类群(Ⅰ—Ⅲ),第Ⅱ类群又被分成5个亚群(Ⅱa—e);共线性分析发现,伴矿景天WRKY基因家族和拟南芥(Arabidopsis thaliana)之间存在7个共线基因对,和玉吊钟(Kalanchoe fedtschenkoi)之间存在53个共线基因对;SpWRKYs基因之间存在19个片段复制基因对;启动子序列分析发现,SpWRKYs启动子有多种与激素和逆境响应等相关的顺式调控元件;镉胁迫表达分析发现,7个...     

甘蓝型油菜BnbHLH92基因的克隆与表达分析

为研究甘蓝型油菜bHLH转录因子的功能,采用同源克隆技术从甘蓝型油菜中克隆了5个BnbHLH92基因全长cDNA序列,分别命名为BnbHLH92-1、BnbHLH92-2、BnbHLH92-3、BnbHLH92-4、BnbHLH92-5,其编码区长度分别为738,657,684,741,717bp.qRT-PCR实验表明,除BnbHLH92-1外,其它的BnbHLH92基因主要在抽薹期和花期的根中表达,BnbHLH92-1主要在抽薹期和花期的根以及二叶一心期的叶中表达.非生物胁迫显著影响BnbHLH92基因的表达,使其表达量升高.低温胁迫下,BnbHLH92基因分别在胁迫后4、6、6、6、6 h表达量达最高.高温胁迫下,5个BnbHLH92基因分别在胁迫后2、6、6、8、4 h表达量达最高.盐胁迫下,BnbHLH92基因分别在胁迫后6、6、24、24、24 h表达量达最高.在ABA诱导下BnbHLH92基因表达量也有不同程度的增加,分析发现BnbHLH92基因的启动子序列上存在ABA响应元件(ABRE).     

3个编码AP2/EREBP转录因子的基因克隆及其在棉纤维中优势表达的初步分析

从棉花cDNA文库中分离到3个编码AP2/EREBP转录因子的cDNAs序列,命名为GhAP2/EREBP1、GhAP2/EREBP2和GhAP2/EREBP3.利用Real-time RT-PCR对这3个基因表达进行分析,结果表明:GhAPZ/EREBP1和GhAP2/EREBP2在棉花纤维中优势表达,而GhAP2/EREBP3在花药中优势表达,在纤维中表达量也较高.这表明上述3个AP2/EREBP转录因子可能在棉花纤维发育过程中起调控作用.     

柳树NAC基因的克隆与表达模式分析

【目的】研究柳树重要抗逆转录因子基因家族,为揭示柳树抗逆分子机制提供理论依据。【方法】以‘苏柳2345’的转录组测序数据为基础,克隆了柳树NAC家族,并分析其序列结构、组织表达特异性以及不同胁迫条件下的表达模式。【结果】克隆的两个柳树NAC转录因子基因分别命名为SlNAC1和SlNAC2。生物信息学分析表明:SlNAC1序列全长为1 126 bp,编码产物含343个氨基酸残基,为稳定的亲水蛋白,定位于细胞核;SlNAC2序列全长为1 139 bp,编码产物含291个氨基酸残基,为稳定的亲水蛋白,定位于叶绿体。两个基因均具有典型的NAM结构域及A、B、C、D、E 5个亚结构域,还包括共同的LPPG、YPNG 和DEE保守基序及NAC抑制结构域。系统进化树分析显示, SlNAC1基因与木薯亲缘关系最近,SlNAC2基因与茄子亲缘关系最近。定量PCR结果显示SlNAC1和SlNAC2均在叶片表达,根中没有表达,为叶片特异性转录因子。定量PCR结果显示SlNAC1基因在脱落酸(ABA)和赤霉素(GA)处理24 h后显著上调,SlNAC2基因在聚乙二醇(PEG)、ABA和GA胁迫后显著上调。【结论】柳树SlNAC1基因在非生物胁迫下表达较为稳定,受ABA和GA胁迫诱导;SlNAC2受干旱、ABA和GA胁迫诱导,受影响明显高于SlNAC1,不受乙烯剂(ETH)胁迫影响。推测SlNAC1和SlNAC2参与GA、ABA信号传导,可能不参与ETH的信号途径。     

薄荷MhWRKY57基因克隆及响应茉莉酸信号的表达分析

【目的】分析薄荷转录因子MhWRKY57的基因和氨基酸特性、亚细胞定位、转录活性、组织表达及茉莉酸甲酯处理下的基因表达水平,为揭示MhWRKY57响应茉莉酸信号调控薄荷精油合成的分子机制提供理论依据。【方法】 利用薄荷转录组数据,采用RT-PCR方法获得MhWRKY57基因编码序列,利用生物信息学分析其基因和氨基酸特性,烟草叶片瞬时表达系统进行亚细胞定位,酵母双杂交系统分析转录激活活性,实时荧光定量 PCR (qRT-PCR)技术分析MhWRKY57基因在各组织及茉莉酸甲酯诱导下的表达模式。【结果】 获得薄荷MhWRKY57基因的编码序列(CDS),编码276个氨基酸,具有典型的WRKY结构域,含34个磷酸化位点,属于非跨膜亲水蛋白,定位于细胞核且具有转录激活活性。系统进化分析表明,薄荷MhWRKY57与大豆Glyma.01G056800.2.p亲缘关系最近。qRT-PCR分析结果表明,MhWRKY57基因在幼叶、成熟叶、花、茎、根中均表达,在根和叶中均明显受到茉莉酸甲酯诱导表达。【结论】 MhWRKY57是一个响应茉莉酸信号的转录因子,可能参与茉莉酸信号介导调控的薄荷精油合成过程。     

菠菜转录因子MYB基因家族的鉴定及表达分析

利用生物信息学手段鉴定了76个具有典型R结构的菠菜转录因子(Spinacia oleracea)MYB,其中包括72条R2R3-MYB基因(2R-MYB)和4条R1R2R3-MYB基因(3R-MYB)。通过生物信息学对菠菜MYB转录因子家族成员的理化性质、染色体定位、结构域序列保守性和系统进化关系进行了分析,结果表明:菠菜MYB家族有32个基因位于染色体正链,另外44个基因位于染色体反链;MYB的DNA结合域中的保守域主要位于两个R重复序列的第二和第三螺旋之间,结合域中每个R重复的第一和第二色氨酸之间的氨基酸序列相对不保守;根据菠菜、拟南芥及甜菜的MYB家族系统进化关系可以推测,菠菜MYB家族中56个成员可以按功能划分为4类,在菠菜的生长发育过程中可能起着重要的调节作用,其余成员中有7个MYB基因可能参与菠菜响应氮素浓度的氮素利用及生长发育进程。     

阳春砂磷酸甲羟戊酸激酶的基因克隆与表达谱分析

目的:为了了解阳春砂磷酸甲羟戊酸激酶(PMK)的功能与表达特性,根据阳春砂的转录组注释设计引物,PCR克隆阳春砂甲羟戊酸途径中的关键酶磷酸甲羟戊酸激酶基因的编码区c DNA,使用生物信息学方法对其编码的蛋白进行相似性检索及同源序列比对,构建进化树,预测二级及三级结构并分析其功能,探索其各器官中的表达差异.结果:获得了全长1 539 bp的编码阳春砂PMK的c DNA序列,命名为Av PMK(Gen Bank登录号:KF569902),编码由512个氨基酸组成的磷酸甲羟戊酸激酶,该蛋白含有GHMP激酶超家族特异的N-保守区域、C-保守区域和ATP结合位点Gly-X-Gly-XX-Ala.Av PMK与其他植物的PMK氨基酸相似性达61%~69%,进化树结果显示其与二穗短柄草(Brachypodium distachyon)等单子叶植物具有较近的亲缘关系.Av PMK蛋白包含14个α-螺旋(Alpha helix),占37.1%;16个β链(Beta strand),占15.6%;32个无规则卷曲结构,占47.3%,无跨膜结构域,其3级结构含有一个催化反应的口袋状结构域.实时荧光定量PCR结果显示Av PMK基因在叶中表达量较高,茎、根中表达量相对较低.结论:首次从阳春砂仁中扩增磷酸甲羟戊酸激酶的基因片段,为分析其基因表达特性及其在砂仁萜类生物合成中的功能奠定基础.     

野桑蚕漆酶基因克隆、序列分析及转录表达谱研究

利用RT-PCR方法,克隆了野桑蚕Bombyx mandarina漆酶基因,获得了其cDNA序列.该序列长2 317bp,含有一个2 295bp的完整开放阅读框,有8个外显子,7个内含子,编码一个由764个氨基酸残基组成的蛋白质,其蛋白质的分子量和等电点分别为84 340.91和6.61.推导的氨基酸序列与其它鳞翅目昆虫(Laccase)基因相应氨基酸序列有较高的同源性,该序列具有它们的漆酶基因所共有的典型特征.组织特异性表达分析表明了该基因仅在野桑蚕的表皮、头部、中肠和血液中有表达.这些结果为进一步研究野桑蚕漆酶基因的功能提供了分子基础.     

Cloning, sequencing and expression analysis of cDNA encoding a constitutive heat shock protein 70 （HSC70） in Fenneropenaeus chinensis

The cDNA encoding hsc70 of Chinese shrimp Fenneropenaeus chinensis was cloned from hepatopancreas by RT-PCR based on its EST sequence. The full length cDNA of 2090 bp contained an open reading frame of 1956 nucleotides and partial 5‘- and 3‘-untranslated region(5‘- and 3‘-UTR). PCR amplification and sequencing analysis showed the existence of introns in the region of 1--547 bp, but they did not exist in the region of 548--2090 bp of hsc70 cDNA. When the deduced 652 amino acid sequence of HSC70 was compared with the members of HSP70 family from other organisms, the results showed 85.9% similarity with HSC71 from Oncorhynchus mykiss and HSC70 from Homo sapiens. It also exhibited 85.8% similarity with HSP70 from Mus musculu and 85.4% with HSC70 from Manduca sexta. Expression analysis showed that hsc70 mRNA was espressed constitutively in hepatopancreas, muscle, eyestalks, haemocytes, heart, ovary, intestine and gills in Fenneropenaeus chinensis. No difference could be detected on hsc70 mRNA level in muscle between heat-shocked and control animals.     

大肠杆菌pheA与tyrB基因的克隆与串联表达

为探讨用基因工程的手段改良苯丙氨酸的发酵菌株，采用聚合酶链反应（ＰＣＲ）的方法，从大肠杆菌总ＤＮＡ中克隆得到了编码苯丙氨酸合成途中的两个关键酶基因－即分枝酸变位酶（ＣＭ）／预苯酸脱水酶（ＰＤ）基因ｐｈｅＡ与苯丙氨酸转氨酶（ＰＡＴ）基因ｔｙｒＢ，在大肠杆菌中进行了这两个基因的单个和串联表达。ｐｈｅＡ和ｔｙｒＢ基因分别都能在λ噬菌体的ＰＲ启动子之后得到较大量的表达，在ＳＤＳ－ＰＡＧＥ上出现清晰的条带，     

大豆bHLH转录因子家族成员的进化及功能分化研究

碱性螺旋-环-螺旋蛋白(basic Helix-Loop-Hleix,bHLH)转录因子家族是动植物中最大的转录因子家族之一,主要由碱性氨基酸区域和螺旋-环-螺旋区域组成,在动植物生长发育和胁迫应答反应中发挥着重要作用.本研究通过对大豆全基因组生物信息学分析和分子生物学研究手段,深入研究了大豆bHLH基因家族的进化机制,同时探讨了该基因家族在大豆中的功能分化.结果表明,大豆中含有340个非冗余的bHLH基因家族成员,通过对这些成员的Pfam蛋白结构域、Motif组成和系统进化关系的分析,将这些成员分为15组共24个亚家族,在大豆20条染色体上呈现不均匀分布.bHLH理化性质差异较大,编码的大豆氨基酸数量为91～815aa,相对分子量为10 273.88～91 300.38,理论等电点为4.56～10.40;碱性氨基酸区含有His5-Glu9-Arg13保守序列,与靶基因结合有关,HLH区含有Arg23和Arg55,与形成二聚体有关,同时含有5种保守元件.多数成员在大豆根以及花发育的5个时期中具有组织表达特异性,不同基因表达量差异较大.     

抗CPAα毒素单链抗体基因的克隆和表达及其生物学特性研究

应用RT—PCR技术 ,从分泌具有中和活性的抗A型产气荚膜梭菌 (CPA)α毒素单克隆抗体的杂交瘤细胞中 ,扩增出单链抗体 (ScFv)基因 ,并将其定向克隆于表达载体 pHOG2 1中 ,构建重组表达载体 pHOG 2E3,转化至大肠杆菌XL1 Blue中 ,筛选出表达菌株XL1 Blue(pHOG 2E3)。SDS PAGE分析结果表明 ,在 2 0℃用IPTG诱导培养时 ,表达的ScFv蛋白占菌体总蛋白的 2 5 %,ScFv蛋白主要以包涵体的形式存在 ,但在胞周质和培养上清中也能检测到ScFv蛋白 ,其中在胞周质中表达的ScFv蛋白占菌体可溶性蛋白的 4 %。生物学试验结果表明 ,ScFv基因表达产物不仅能够中和α毒素的磷酯酶C活性 ,而且对攻击致死剂量α毒素的小鼠产生良好的被动保护作用