麻疯树curcin 启动子的分离及其在转基因烟草中的功能分析

核糖体失活蛋白是一类可以使核糖体28S rRNA的保守a茎环结构域脱嘌呤的蛋白质。麻疯树毒蛋白(curcin)前体基因编码麻疯树胚乳I 型核糖体失活蛋白。从麻疯树基因组中克隆得到其5'侧翼区0.6 kb长的片段, 将该片段插入pBI121载体置换其中的CaMV 35S启动子并在相应的转基因烟草中检测报告基因GUS的表达情况。经过GUS活性检测分析发现, 该0.6 kb长的片段能够启动报告基因在种子中的表达, 并且其在种子不同发育阶段的表达活性存在差异。同时, GUS组织化学染色定位分析表明, 在双子叶植物中该启动子片段是胚乳特异性表达的, 它从心形胚时期开始持续地在烟草的胚乳中发挥启动活性。     

麻疯树逆境蛋白(curcin 2)基因在烟草中的表达

麻疯树（Jatropha curcas）幼苗在干旱、高低温胁迫和真菌浸染下,其叶片中诱导产生了一种新的毒蛋白curcin 2。这意味着curcin 2在其它植物中的异源表达可能会增强植物对外界胁迫的抵抗。curcin 2 cDNA的两个片断：cur2p片断（编码前成熟蛋白）和cur2m片断（编码成熟蛋白）,通过农杆菌的介导分别转化烟草并获得转基因植株。但是,只有在插入了cur2p片断的烟草中检测到了curcin 2蛋白的表达。同时,curcin 2在烟草中的表达增强了植株对烟草花叶病毒（TMV）的抗性。     

麻疯树核糖体失活蛋白基因的克隆和表达

麻疯树(Jatropha curcas L.)核糖体失活蛋白(curcin)是存在于麻疯树种子中的一种毒性较强的蛋白,它与蓖麻毒蛋白和相思子毒蛋白的性质相似,属Ⅰ型核糖体失活蛋白。从麻疯树种子中分离得到一种分子量为28.2kD的蛋白质,其对无细胞系统中蛋白质合成的抑制活性较强,IC_(50)为(0.19±0.01)nmol/L,具有RNA N-糖苷酶活性。依据curcin的N端部分氨基酸设计简并引物,通过RT-PCR和5′-RACE技术从未成熟种子总RNA中克隆到curcin全长cDNA序列。该cDNA全长由1 173个碱基组成,包含一个编码293个氨基酸的前体蛋白,前42个氨基酸为信号肽。推测的多肽序列与测定的蛋白质N端序列相同,与多种已发表的Ⅰ型核糖体失活蛋白和Ⅱ型核糖体失活蛋白的A链有一定的同源性。将curcin的编码区与表达载体pQE-30相连后,转入大肠杆菌(Escherichia coil)M15菌株中得到了有效的表达。将表达的融合蛋白纯化后发现,它具有抑制无细胞系统蛋白质合成的能力。     

麻疯树核糖体失活蛋白基因的克隆和表达

麻疯树(Jatropha curcas L.)核糖体失活蛋白(curcin)是存在于麻疯树种子中的一种毒性较强的蛋白,它与蓖麻毒蛋白和相思子毒蛋白的性质相似,属Ⅰ型核糖体失活蛋白.从麻疯树种子中分离得到一种分子量为28.2 kD的蛋白质,其对无细胞系统中蛋白质合成的抑制活性较强,IC50为(0.19±0.01)nmol/L,具有RNA N-糖苷酶活性.依据curcin的N端部分氨基酸设计简并引物,通过RT-PCR和5'-RACE技术从未成熟种子总RNA中克隆到curcin全长cDNA序列.该cDNA全长由1 173个碱基组成,包含一个编码293个氨基酸的前体蛋白,前42个氨基酸为信号肽.推测的多肽序列与测定的蛋白质N端序列相同,与多种己发表的Ⅰ型核糖体失活蛋白和Ⅱ型核糖体失活蛋白的A链有一定的同源性.将curcin的编码区与表达载体pQE-30相连后,转入大肠杆菌(Escherichia coil)M15菌株中得到了有效的表达.将表达的融合蛋白纯化后发现,它具有抑制无细胞系统蛋白质合成的能力.     

麻疯树核糖体失活蛋白Curcin2的原核可溶性表达及其抗真菌活性研究

Curcin2是麻疯树幼苗在真菌侵染、干旱及高低温胁迫下诱导产生的一种核糖体失活蛋白.采用分子克隆的方法,从麻疯树基因组中扩增到Curcin2成熟肽编码基因,分别连接到质粒载体pGEX-6p-1、pMAL-c5E上,转化大肠杆菌最终获得重组菌PGC(pGEX-6p-1-curcin2)和PMC(pMAL-c5E-curcin2).在不同温度、IPTG浓度、时间诱导下,Curcin2在重组菌PGC中均以包涵体形式表达,在重组菌PMC中主要以可溶性融合蛋白形式表达,且蛋白质的表达量与诱导条件相关.重组菌PMC表达可溶性curcin2的优化条件为:温度28℃,IPTG 0.3mmol/L,时间8h,此条件下目的蛋白占总蛋白表达量的30.6％,1L培养物中可获得19.74mg电泳纯的重组蛋白.重组蛋白可被MBP TrapTM HP柱亲和纯化并与curcin抗体发生抗原抗体反应.体外抗真菌活性实验表明,纯化后的curcin2融合蛋白有抑制真菌生长作用,且对小麦赤霉、油菜菌核的抑制作用强于curcin.此蛋白的获得为其相关功能的研究奠定了基础.     

麻疯树毒蛋白curcin2基因的两个原核表达载体的构建和表达

从麻疯树基因组中扩增到毒蛋白curcin2基因成熟肽编码区,成功构建了原核表达载体pET-C和pQE-C,并转化大肠杆菌获得转化子PCB和PCM。在不同温度、不同浓度IPTG和不同诱导时间下,PCB都没有curcin2重组蛋白产生,而PCM能表达出curcin2,主要以包涵体形式存在。诱导温度较低时,延长诱导时间有可溶性的curcin2产生。在文章的实验中,PCM表达curcin2的优化条件为:温度16℃,IPTG1mmol·L-1,时间16￣24h。     

麻疯树两种核糖体失活蛋白的表达模式分析

目前,麻疯树核糖体失活蛋白已发现2种:种子中的毒蛋白curcun和叶片中的逆境诱导蛋白curcin-L,其基因的表达模式均呈现出器官特异性.mRNA和蛋白质水平分析显示,curcin只在种子的胚乳中积累,且表达开始于心型胚时期,成熟时达到最高;而curcin-L在正常生理条件下并不表达,但受脱落酸、水杨酸、干旱,高低温等胁迫诱导时仅在叶片中表达.     

麻疯树MIPS基因启动子的分离及在烟草原生质体中瞬时表达活性分析

根据麻疯树MIPS基因序列,设计特异性的巢式引物,运用TAIL-PCR法两次步移得到MIPS基因5＇端侧翼序列,序列分析显示含有多个胁迫应答相关元件,如ABRE、HSE等。以该序列为基础,PCR扩增得到5个5＇端不同长度的缺失片段,分别插入pBI221载体置换CaMV35S启动子,构建的表达载体在PEG介导下转入烟草叶片原生质体进行瞬时表达,检测GUS报告基因的活性。经GUS活性荧光定量检测发现,分离到的MIPS基因侧翼序列5＇端不同缺失片段都能启动GUS报告基因表达,启动活性最高的是WQ1区（-565bp）,核心区位于-565～-449bp。在100μmol·L-1ABA诱导下启动活性增强,但不同区段的增长幅度不同。WQ1区增长幅度最大,比未处理时提高41.4%。     

麻疯树的二萜成分

从麻疯树（Jatropha curcas L.)的根中分离得到3个二萜,鉴定为麻疯树酚酮A（iatropholone A)。麻疯树酚酮B（jatropholone B)和一个新的二萜,其结构推定为16－羟基麻疯树酚酮（16-hydroxyjatropholonc),定名麻疯树醇（jatrophol),同时还得到麻疯素（jatrophin),β-谷甾醇－β-D－葡萄糖甙（β－D－glucoside-β-sitosterol),tomentin,蒲公英脑（taraxerol)以及β-谷甾醇。     

麻疯树毒素的分离及其某些性质

用磷酸盐缓冲液萃取,硫酸铵盐析及葡聚糖凝胶G-100柱层析后,从麻疯树种子分离出三个蛋白组分。其表观分子量分别约为34000、27000和9500道尔顿;等电点分别为8.1、8.8和8.8.它们在紫外区呈现典型的蛋白吸收光谱。组分Ⅰ毒性最大,小鼠腹腔注射半致死量(LD₅₀)为6.39mg。     

小桐子低温诱导型启动子JcDnaJ20p的克隆及烟草转化功能鉴定

低温转录组数据显示,DnaJ20是小桐子低温诱导基因.为鉴定该基因启动子的低温诱导活性,基于PCR技术从小桐子叶片基因组DNA中克隆到DnaJ20基因(JcDna20)启动子序列,命名为JcDnaJ20p,利用重组技术构建了JcDnaJ20p启动子驱动GUS标记基因的植物双元表达载体pCambia1381Z-JcDna...     

Isolation and Characterization of a Curcin Promoter from <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. and Its Regulation of Gene Expression in Transgenic Tobacco Plants

Ribosome-inactivating proteins (RIPs) represent those proteins that universally depurinate conserved α-sarcin loops of large rRNAs. In this study, a 0.6-kb fragment of a 5′ flanking region preceding a curcin gene, encoding a type I RIP curcin, of Jatropha curcas L. endosperm was cloned, and its regulation of expression of the β-glucuronidase (GUS) reporter gene was investigated in transgenic tobacco. Analysis of GUS activities showed that the 0.6-kb flanking fragment of the curcin gene was sufficient to drive the GUS reporter gene expression in tobacco seed. The activity of this flanking fragment was analyzed at different stages of seed development. Histochemical localization of GUS activity indicated that the promoter was specifically active in the endosperm tissue of the dicotyledonous tobacco embryo. Moreover, this activity was first initiated at the heart-shaped embryonic stage during seed development.     

The Expression of a Cytosolic Cyclophilin Promoter from Periwinkle in Transgenic Tobacco Plants

The cloning of a 465 bp fragment from the 5-flanking region of the gene encoding a cytosolic cyclophilin from periwinkle was achieved through inverse polymerase chain reaction. The DNA fragment was fused to a gusA-intron marker then introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Histochemical analysis of the transgenic shoot cultures demonstrated that the construct was able to drive GUS expression in stomata guard cells, but not in mesophyll cells when shoots were still attached to the callus from which they were initiated. In separated transgenic shoots and in seedlings, GUS was expressed in external and internal phloem and root hairs, respectively. GUS activity in transgenic tobacco seedlings was also investigated by fluorimetric assays. Treatments with NaCl or ABA decreased promoter activity whereas treatment with yeast extracts increased it.     

Isolation and identification of miRNAs in Jatropha curcas

MicroRNAs (miRNAs) are small noncoding RNAs that play crucial regulatory roles by targeting mRNAs for silencing. To identify miRNAs in Jatropha curcas L, a bioenergy crop, cDNA clones from two small RNA libraries of leaves and seeds were sequenced and analyzed using bioinformatic tools. Fifty-two putative miRNAs were found from the two libraries, among them six were identical to known miRNAs and 46 were novel. Differential expression patterns of 15 miRNAs in root, stem, leave, fruit and seed were detected using quantitative real-time PCR. Ten miRNAs were highly expressed in fruit or seed, implying that they may be involved in seed development or fatty acids synthesis in seed. Moreover, 28 targets of the isolated miRNAs were predicted from a jatropha cDNA library database. The miRNA target genes were predicted to encode a broad range of proteins. Sixteen targets had clear BLASTX hits to the Uniprot database and were associated with genes belonging to the three major gene ontology categories of biological process, cellular component, and molecular function. Four targets were identified for JcumiR004. By silencing JcumiR004 primary miRNA, expressions of the four target genes were up-regulated and oil composition were modulated significantly, indicating diverse functions of JcumiR004.     

玉米茎特异启动子克隆及其在转基因烟草中的活性分析

从玉米自交系‘综31’基因组中分离了1个茎特异表达启动子,命名为ZmSSP。用ZmSSP替换植物表达载体pCAMBIA3301的CaMV35S启动子,构建了ZmSSP驱动GUS报告基因的重组表达载体pCAMBIA3301-ZmSSP-GUS,并采用农杆菌介导法转化烟草,对转基因烟草营养器官中GUS表达模式进行了分析。结果表明:在烟草中ZmSSP活性低于CaMV35S启动子;不同转基因株系中ZmSSP活性及模式有显著差异;10个转基因株系统计结果表明,GUS表达量最高的营养器官是叶柄,平均是Actin基因表达量的2.71倍;其次是叶片和茎,在根中的GUS表达量最低,平均是Actin基因表达量的29.6%,是叶柄中活性的10.9%。研究认为,ZmSSP是较好的组织特异性启动子,适用于通过植物基因工程技术驱动目的基因进行地上营养器官的性状改良。