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1.
桃PpMADS1基因启动子的克隆及功能分析   总被引:3,自引:0,他引:3  
PpMADS1基因属于一类MADS box 基因,在植物的花发育调控中起着重要的作用。通过Genome Walking的方法从桃基因组中分离了长度为1 814bp的PpMADS1基因启动子片段,序列分析表明,在此启动子上不仅含有TATA box 和CAAT box基本元件,而且含有大量的与光调节有关的调控元件,如GT-1,Sp1和as-2-box,另外存在两个CArG-box元件、一个G-box元件和一个TGA-element,说明该启动子可能受光周期和激素的调控。将该启动子通过5′端缺失,分区段与GUS报告基因连接构建表达载体,并转化拟南芥。GUS组织化学染色分析结果表明,在-197到-454bp有促使GUS在花原基中表达的花原基特异性元件,在-454到-678bp之间存在促使GUS在萼片和花瓣表达的特异性元件,在-678到-978bp存在负调控作用元件,阻遏了GUS基因在花药中的表达。  相似文献   

2.
桃中两个MADS box基因的克隆与表达分析   总被引:7,自引:1,他引:6  
为研究李属(Prunus sp.)果树生殖调控的相关基因,对国际公共数据库中的李属植物的EST(expressed sequence tags)序列进行了电子拼接,获得了8个MADS box基因的cDNA序列,并利用PCR技术从桃中克隆出其中的两个cDNA,分别命名为PpMDS4和PpMADS6,在GenBank中的登录号为AY705972和AY705973。PpMADS4基因长850bp,包含一个732bp的开放阅读框,编码243个氨基酸。PpMADS6基因长1190bp,包含1个768bp的开放阅读框,编码256个氨基酸。PpMADS4和PpMADS6在序列上分别与拟南芥中的AGAMOUS基因和矮牵牛中的PFG基因高度同源。RT-PCR分析表明,PpMADS4基因在桃的花瓣、心皮、果实及果仁中表达,应属于控制花器官发育的C类MADS box基因。PpMADS6基因在桃的叶、萼片、花瓣、心皮及果实中表达,应属于调控植物由营养生长向生殖生长过渡的A类MADSbox基因。  相似文献   

3.
采用PCR技术从拟南芥中克隆了SCBP60g基因的启动子,并与GUS报告基因融合构建重组表达载体,转化野生型拟南芥,对获得的转基因株系进行GUS组织染色,从基因调控水平上探讨其在功能方面的差异。结果显示:SCBP60g基因的启动子能指导GUS报告基因在拟南芥的根、茎、叶和花中表达,并且在这些部位的维管束表达较强。这种表达方式与LCBP60g基因的启动子指导的GUS基因组织化学染色有差异,表明这个启动子的表达调控具有一定的特异性。  相似文献   

4.
为研究拟南芥甲基结合蛋白基因AtMBP11在种子形成和萌发过程中的调控模式,克隆拟南芥AtMBP11启动子,将其替换植物表达载体pBI121的35S启动子序列,转入拟南芥基因组中.转基因拟南芥后代卡那霉素抗性发生分离,选取具有3∶1分离比的后代自交,产生纯合的具有单拷贝插入的后代.转基因后代GUS染色结果表明,新克隆的MBP启动子控制基因在种子、花药和花粉中高效表达.通过对AtMBP11核心启动子缺失分析表明,G-box元件是主要功能元件.  相似文献   

5.
NAC转录因子家族是植物中特有的、家族数目较多的一类转录因子家族,对植物生长发育起重要作用。了解CiNAC038的表达调控分子机制,为中间锦鸡儿CiNAC038功能研究奠定基础。以中间锦鸡儿为植物材料,通过染色体步移法克隆启动子序列,并对启动子序列上的响应元件进行分析。构建GUS表达载体,并转化拟南芥,对拟南芥的组织特异性进行表达分析,用ABA诱导转基因植株,研究ABA与CiNAC038的关系。结果显示,克隆了1 800 bp的CiNAC038启动子序列,该启动子包含多种顺式元件。成功构建植物表达载体ProCiNAC038∶GUS,通过浸花法转化至野生型拟南芥。GUS组织化学染色结果显示,转基因拟南芥幼苗根部染色较深,胚轴无染色;成熟期转基因拟南芥的叶脉、果荚两端、花瓣、花药等组织染色较深,茎无染色。CiNAC038启动子驱动的GUS报告基因主要在植物叶片、根和花的组织器官表达。进一步ABA诱导表达分析发现,GUS染色随着浓度增加颜色越浅。CiNAC038启动子是ABA抑制型启动子。  相似文献   

6.
绿色组织特异表达启动子可调控外源基因只在受体作物的绿色组织中定点、高效地表达。以普通野生稻为实验材料,克隆了绿色组织特异表达启动子Or GSP,构建Or GSP和GUS基因融合的表达载体,转入拟南芥中鉴定功能。启动子Or GSP长度为825 bp,含有基本的转录起始元件TATA-box和CAAT-box,以及光响应元件TCCC-motif、Sp1、G-box、I-box、GA-motif和as-2-box等。转基因拟南芥GUS组织化学染色结果表明,启动子Or GSP调控GUS基因只在绿色组织中特异表达。GUS活性测定结果显示,叶和茎中的GUS活性比根中明显提高。普通野生稻中克隆的启动子Or GSP为绿色组织特异表达启动子,可为作物分子育种提供新的调控元件。  相似文献   

7.
毛白杨PtSEP3-1基因启动子的克隆分析及其表达载体构建   总被引:2,自引:0,他引:2  
SEP(SEPALLATA)类基因属于花器官发育ABCDE模型中的E类基因,拟南芥中的研究表明该类基因可能具有控制花器官形态发育以及激活其它类型基因的功能,是一类花发育过程中的关键基因。因此,研究杨树SEP类基因启动子表达特性对于杨树的开花调控研究具有重要意义。本文根据毛白杨SEP3基因和毛果杨基因组序列设计引物,通过PCR获得了PtSEP3-1基因上游2000bp的序列。序列分析结果表明该序列具有启动子的基本元件TATA-box和CAAT-box,还包含大量光响应元件ACE、Box I和Box4等,此外还有脱落酸响应元件ABRE,赤霉素响应元件GARE-motif以及胁迫响应元件HSE、TC-richrepeats等。进一步构建了一个以PtSEP3-1启动子驱动GUS基因的植物表达载体pPtSEP3-1protest,为该启动子的功能鉴定奠定了基础。  相似文献   

8.
拟南芥AtNCED2基因启动子区域序列克隆及其活性分析   总被引:1,自引:0,他引:1  
目的:克隆拟南芥AtNCED2基因启动子区域序列,并分析其组织器官特异性及对外界刺激的响应.方法:通过PCR从拟南芥基因组中克隆AtNCED2基因5'侧翼2295bp启动子区域序列(AtNCED2p),并进行生物信息学分析.构建AtNCED2p驱动GUS的植物双元表达载体pAtNCED2p::GUS,通过根癌农杆菌介导法将其转化野生型拟南芥,检测转基因植侏中GUS表达的组织器官特异性.结果:该启动子序列中存在TATA-box、CAAT-box、根器官特异性元件、ABA响应元件、低温响应元件、昼夜节律响应元件等顺式作用元件.GUS活性主要集中在转基因拟南芥根尖及侧根发生部位.外源ABA处理的转基因植株根中GUS活性为174.8nmol 4-MU min-1 mg-1蛋白,明显高于对照值91.7nmol 4-MU min-1mg-1蛋白.结论:AtNCED2基因可能在根的生长和发育中起作用,且外源ABA处理增强其在根中的表达.  相似文献   

9.
为探明盐穗木盐相关转录因子基因Hc SCL13的表达调控规律,利用基因组步移法成功克隆获得该基因2 200 bp的启动子序列。Plant CARE数据库分析结果表明,该启动子不仅含有启动子区的核心元件CAAT-box和TATA-box,还包含多个与逆境应答有关的顺式调控元件。将克隆获得的Hc SCL13转录因子基因启动子序列定向替换p BI121载体上的35S启动子,构建融合表达载体并转染模式植物拟南芥,对转基因拟南芥进行GUS组织化学染色。结果显示转基因拟南芥整株被染色,提示该启动子具有表达活性且可能为组成型启动子。  相似文献   

10.
Col生态型拟南芥AP3基因启动子克隆及植物表达载体构建   总被引:1,自引:0,他引:1  
隶属于MADS-Box基因家族的拟南芥花器官B类特征基因APETALA3 (AP3)在花瓣和雄蕊中特异性地表达;AP3基因编码转录因子,与A类和C类特征基因协同作用控制双子叶植物花瓣和雄蕊的发育.研究表明AP3基因启动子为花特异表达启动子.因此,AP3基因启动子的克隆及功能鉴定对于园林植物与花相关的商业性状的定向改良具有重要作用.本文根据GenBank数据库报道的Ler生态型拟南芥(Arabido-psis thaliana) AP3基因启动子序列(U30729)设计了一对特异性扩增引物,基于PCR技术,用高保真的KOD-plus DNA聚合酶扩增了长度为1 767 bp的Col生态型拟南芥AP3基因启动子,并命名为pAtAP3,其GenBank登录号为FJ619533.Bl2seq在线分析表明pAtAP3与U30729序列的相似性达98%,与Col生态型拟南芥BAC克隆T12E18 (AL132971) 9 264~11 030之间的碱基序列相似性达100%,且该段序列的下游基因编码AP3蛋白(CAB81799),说明克隆序列为Col生态型拟南芥AP3基因的启动子.PLACE在线分析表明pAtAP3具有基本的启动子元件TATA-box和CAAT-box,还包含大量与花特异表达相关的顺式元件CArG1、CArG2、CArG3和anther-box等.本试验进一步构建了植物表达载体pAtAP3::GUS,为该启动子的功能鉴定奠定了基础.  相似文献   

11.
本研究克隆了甘蓝型油菜线粒体功能未知基因 ORF 117,构建了带有线粒体转运信号肽序列(TP )的植物过表达载体35S ∷TP-ORF117、35S ∷TP-EGFP 和35S ∷TP-ORF117-EGFP ,转化野生型拟南芥并进行抗除草剂筛选,共获得纯合的转基因拟南芥株系62个。qPCR 表明,ORF 117在转基因材料中得到高效过表达。用转35S ∷TP-EGFP 、35S ∷TP-ORF117-EGFP 拟南芥制备原生质体,经线粒体特异染料染色,在激光共聚焦显微镜下做线粒体、GFP 共定位检测,GFP 蛋白和 ORF117-EGFP 融合蛋白被准确定位到了线粒体。  相似文献   

12.
PpMADS1, a member of the euAP1 clade of the class A genes, was previously cloned from peach. In this study, PpMADS1 was constitutively expressed in Arabidopsis thaliana to study its function in plant development. The transgenic A. thaliana plants containing 35S::PpMADS1 showed severe phenotype variation including early flowering, conversion of inflorescence branches to solitary flowers, formation of terminal flowers, production of higher number of carpels, petals, and stamens than non-transgenic plants, and prevention of pod shatter. Significantly, the transgenic plants produced more than one silique from a single flower. The results obtained by using cDNA microarray and real-time PCR analyses in the transgenic Arabidopsis indicated that PpMADS1 might play dual roles in regulating the floral meristem development by activating or repressing different sets of genes that would determine the different fate of a floral meristem. In addition, the PpMADS1 gene promoter was further cloned, and deletion analyses were conducted by using fused GUS as a reporter gene in transgenic A. thaliana. Histochemical staining of different organs from transgenic plants revealed the region between ?197 and ?454?bp was specific for GUS expression in flower primordium, and the region between ?454 and ?678?bp was specific for GUS expression in sepals and petals. In contrast, a negative regulatory element present between ?678 and ?978?bp could suppress GUS expression in filament.  相似文献   

13.
We cloned 10 Japanese pear (Pyrus pyrifolia) MIKC-type II MADS-box genes, and analyzed their expression during fruit development and ripening. PpMADS2-1 was APETALA (AP)1-like; PpMADS3-1 was FRUITFULL (FUL)/SQUAMOSA (SQUA)-like; PpMADS4-1 was AGAMOUS-like (AGL)6; PpMADS5-1 and PpMADS8-1 were SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC)-like; PpMADS9-1, PpMADS12-1, PpMADS14-1 and PpMADS16-1 were SEPALLATA (SEP)-like; while PpMADS15-1 was AGL/SHATTERPROOF (SHP)-like. Phylogenetic analysis showed their grouping into five major clades (and 10 sub-clades) that was consistent with their diverse functional types. Expression analysis in flower tissue revealed their distinct putative homeotic functional classes: A-class (PpMADS2-1, PpMADS3-1, PpMADS4-1, and PpMADS14-1), C-class (PpMADS15-1), E-class (PpMADS9-1, PpMADS12-1, and PpMADS16-1) and E (F)-class (PpMADS5-1 and PpMADS8-1). Differential gene expression was observed in different fruit tissues (skin, cortex and core) as well as in the cortex during the course of fruit development and ripening. Collectively, our results suggest their involvement in the diverse aspects of plant development including flower development and the course of fruit development and ripening.  相似文献   

14.
15.
MADS box proteins play an important role in floral development. To find genes involved in the floral transition of Prunus species, cDNAs for two MADS box genes, PpMADS1 and PpMADS10, were cloned using degenerate primers and 5'- and 3'- RACE based on the sequence database of P. persica and P. dulcis. The full length of PpMADS1 eDNA is 1, 071bp containing an open reading frame (ORF) of 717bp and coding for a polypeptide of 238 amino acid residues. The full length of PpMADS10 cDNA is 937bp containing an ORF of 633bp and coding for a polypeptide of 210 amino acid residues. Sequence comparison revealed that PpMADS1 and PpMADS10 were highly homologous to genes AP1 and PI in Arabidopsis, respectively. Phylogenetic analysis indicated that PpMADS1 belongs to the euAP1 clade of class A, and PpMADS10 is a member of GLO/PI clade of class B. RT-PCR analysis showed that PpMADS1 was expressed in sepal, petal, carpel, and fruit, which was slightly different from the expression pattern of AP1; PpMADS10 was expressed in petal and stamen, which shared the same expression pattern as PI. Using selective mapping strategy, PpMADS1 was assigned onto the Bin 1:50 on the G1 linkage group between the markers MCO44 and TSA2, and PpMADS10 onto the Bin 1:73 on the same linkage group between the markers Lap-1 and FGA8. Our results provided the basis for further dissection of the two MADS box gene function.  相似文献   

16.
In Arabidopsis thaliana, cis-regulatory sequences of the floral homeotic gene AGAMOUS (AG) are located in the second intron. This 3-kb intron contains binding sites for two direct activators of AG, LEAFY (LFY) and WUSCHEL (WUS), along with other putative regulatory elements. We have used phylogenetic footprinting and the related technique of phylogenetic shadowing to identify putative cis-regulatory elements in this intron. Among 29 Brassicaceae species, several other motifs, but not the LFY and WUS binding sites identified previously, are largely invariant. Using reporter gene analyses, we tested six of these motifs and found that they are all functionally important for the activity of AG regulatory sequences in A. thaliana. Although there is little obvious sequence similarity outside the Brassicaceae, the intron from cucumber AG has at least partial activity in A. thaliana. Our studies underscore the value of the comparative approach as a tool that complements gene-by-gene promoter dissection but also demonstrate that sequence-based studies alone are insufficient for a complete identification of cis-regulatory sites.  相似文献   

17.
水稻谷氨酰半胱氨酸合成酶基因的结构和表达分析   总被引:6,自引:0,他引:6  
利用该实验室T-DNA标签的编号为L395的水稻突变体,克隆了一个编码水稻谷胱苷肽(GSH)合成途径中关键酶即谷氨酰半胱氨酸合成酶(GCs)的基因,将其命名为OsGCS(Genbank accession No.AJ508915).该基因位于水稻第五染色体上,OsGCS基因含有15个外显子和14个内含子,编码492个氨基酸.该基因与拟南芥的GCS基因相比较,编码区域同源性较高,而启动子区域的序列没有显著的相似性.通过RT-PCR的方法确定OsGCS基因的转录起始位点可能位于翻译起始位点(ATG)上游211bp处.在L395突变体中,T-DNA是单拷贝形式插入在OsGCS基因的第二内含子和外显子连接处,并且造成了3个碱基的缺失.在重金属耐受性、OsGCS基因表达以及体内GsH含量方面突变体L395和对照中花11之间没有明显的差别.  相似文献   

18.
We examined the degree of conservation of gene order in two plant species, Prunus persica (peach) and Arabidopsis thaliana (thale cress), whose lineages diverged more than 90 million years ago. In the three peach genomic regions studied, segments with a gene order congruent with A. thaliana were short (two to three genes in length); and for any peach region, corresponding segments were found in diverse locations in the A. thaliana genome. At the gene level and lower, the A. thaliana sequence was enormously useful for identifying likely coding regions in peach sequences and in determining their intron-exon structure. The peach BAC sequence data reported here contained a BLAST-detectable putative coding sequence an average of every 7 kb, and the peach introns identified in this study were, on average, almost twice the length of the corresponding introns in A. thaliana.  相似文献   

19.
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