柑橘CitMYB40转录因子的克隆与表达分析

【目的】分析R2R3-MYB家族成员Cit MYB40的生物学功能,为柑橘抗逆基因筛选和抗逆生理机制探索提供依据。【方法】以‘资阳’香橙(Citrus junos‘Ziyang’)为试材,采用生物信息学和基因表达技术,通过不同器官组织比较、植物激素和非生物胁迫处理,研究Cit MYB40的表达规律,并预测其基本生物学功能。【结果】Cit MYB40基因的开放阅读框(ORF)为984 bp,可编码297个氨基酸残基多肽。该基因含有3个内含子、4个外显子,与葡萄Vv MYB39、胡杨Pe MYB86、马铃薯St MYB34、麻风树Jc MYB34等同源蛋白的相似度为45.57%~53.51%。实时定量分析发现,Cit MYB40基因在植株不同组织器官间的表达具有明显差异,在花中的表达量最高,根中最低。在植物激素作用下,叶中的表达量明显高于根中,但ABA、SA、Me JA、ACC均可诱导Cit MYB40基因在根和叶中的表达。在非生物胁迫下,PEG6000、Na Cl和4℃低温均对Cit MYB40的表达有上调作用,且Na Cl处理时,Cit MYB40基因在根和叶中的表达模式与PEG6000处理相似。【结论】柑橘Cit MYB40在不同组织中的表达存在显著差异,并受不同植物激素和非生物胁迫诱导,推测其在柑橘生长发育和逆境响应中起到了一定的作用。     

毛竹EST资源SSR标记分析与筛选

 对来源于NCBI公共数据库的非冗余3 087条毛竹（Phyllostachys edulis）EST序进行简单重复序列SSR搜索发现：在401条毛竹EST序列中共查找到408个SSR位点,平均6.8 kb EST序列中含有1个SSR。其中二核苷酸和三核苷酸重复是毛竹EST-SSR的主要重复类型,分别占SSR总数的43.14%和49.75%。SSR的不同重复基序中,最常见的重复基序是：(AG)_n,占37.01%,其余出现频率较高的依次为(TC)_n、(AGC)_n、(GAG)_n和(CGC)_n。根据搜索结果设计了182条毛竹EST-SSR引物,以12个不同种属的竹子DNA为模板,对引物进行了稳定性、通用性的验证与评价。结果表明有42对引物有较清晰且稳定的目标扩增产物,其中39对引物的产物存在多态性,说明设计开发的毛竹的EST-SSR标记是可行且有效的。     

香蕉延长因子1A（MaEF1A）基因的分离及特征分析

采用筛选香蕉果实采后cDNA文库的方法,首次获得了一条长为1 341 bp的cDNA序列,命名为MaEF1A。生物信息学分析结果表明,它与麝香百合延长因子1A 3的mRNA有84%的同源性,与烟草延长因子1A的mRNA有83%的同源性,推测它可能为编码香蕉延长因子1A（elongation factor-1 alpha...     

香蕉trihelix转录因子MaGTL1a的分离及特性

【目的】研究香蕉MaGTL1a转录因子的特性及其基因表达规律,探讨MaGTL1a转录因子在香蕉果实成熟过程中的作用。【方法】以香蕉果肉c DNA为模板,采用RT-PCR获得MaGTL1a序列;利用烟草瞬时表达法和酵母系统分析MaGTL1a亚细胞定位和转录调控活性;通过实时荧光定量PCR分析MaGTL1a在香蕉果实成熟过程中的表达;运用烟草BY2悬浮细胞瞬时表达法研究MaGTL1a启动子活性。【结果】MaGTL1a c DNA序列含有1个2 283 bp的开放阅读框,编码761个氨基酸,属于trihelix转录因子的GT-2亚家族;亚细胞定位和转录活性分析显示,MaGTL1a定位于细胞核,并在酵母和植物体内具有转录激活活性;实时荧光定量PCR和启动子活性试验表明,MaGTL1a转录水平和启动子活性均受乙烯诱导,并且MaGTL1a转录水平随着香蕉果实的成熟进程而明显增强。【结论】MaGTL1a是一个受乙烯诱导和核定位的转录激活子,可能参与了香蕉果实成熟的调控。     

毛竹纤维素合成酶基因PeCesA的克隆及组织表达谱分析

根据物种间同源基因设计引物,对构建好的毛竹笋全长cDNA文库进行大规模PCR筛选,成功分离出了毛竹纤维素合成酶基因PeCesA的cDNA序列,全长共3 545 bp（GenBank登录号：FJ799713）。该序列包含3 243 bp的开放阅读框（ORF）,编码1 081个氨基酸。通过核酸序列比对发现,该序列与绿竹、水稻、玉米、小麦和大麦的纤维素合成酶基因同源性极高（> 90%）。蛋白序列比对分析发现：PeCesA蛋白含有植物纤维素合成酶特有的保守区、可变区、N端Zn指结构域以及8个跨膜区;系统发育分析表明PeCesA与绿竹BoCesA4、水稻OsCesA4、玉米ZmCesA4属于同一进化分支。运用实时定量PCR法分析PeCesA基因的组织表达特异性发现,PeCesA在毛竹根部的表达量最高,茎部其次,叶中较少;在竹笋基部的表达量远远高于竹笋上部。随着PeCesA基因表达量的增高,组织中的纤维素含量亦相应增高。推测PeCesA参与了毛竹次生细胞壁中纤维素的生物合成。     

Hepatocyte growth factor protects hepatocytes from apoptosis induced by actinomycin D

AIM: To investigate the anti-apoptotic effect of hepatocyte growth factor (HGF) on actinomycin D (ActD)-induced apoptosis in hepatocytes and the possible pathway. METHODS: Hepatocytes were exposed to ActD and HGF. The cytotoxic effects of ActD were tested by MTT. Apoptotic cells were identified by Hoechst 33342 staining, flow cytometry and detection of DNA fragmentation with agarose gel. Akt and phospho-Akt were detected by Western blotting analysis. RESULTS: The results showed that ActD induced apoptosis in hepatocytes 5 h after treatment. Phosphatidylinositol-3 kinase (PI-3K) specific inhibitor wortmannin enhanced the apoptotic effect of ActD. Furthermore, HGF significantly reduced the apoptosis in hepatocytes induced by ActD in a concentration-dependent manner. In the presence of wortmannin, HGF did not overcome apoptosis. CONCLUSION: Wortmannin enhances the apoptotic effect of ActD. HGF protects hepatocytes from apoptosis induced by ActD through a PI3K/Akt pathway.     

Isolation of plant transcription factors using a modified yeast one-hybrid system

Background  

The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontech™ product, the MATCHMAKER Library Construction and Screening Kit 3. This kit employs SMART technology for the amplification of full-length cDNAs, in combination with cloning using homologous recombination.     

湖北海棠植物络合素合酶MhPCS基因克隆及表达分析

【目的】为了探明植物络合素酶参与植物解毒重金属的作用,【方法】以湖北海棠(Malus hupehensis)为试材,克隆其植物络合素合酶基因(MhPCS)编码区序列,并研究该基因的表达特点。【结果】结果表明MhPCS基因编码区全长为1 494 bp,编码497个氨基酸。序列分析表明,MhPCS编码的氨基酸序列由2个典型的植物络合素合酶亚家族结构域组成,具有3个相邻的Cys-Cys元件(331-332、351-352和369-370位氨基酸)和植物络合素合酶蛋白的特征位点(Cys-56、Cys-90/91和Cys-109)。【结论】湖北海棠MhPCS与杜梨PbPCS蛋白同源性最高(94%),属于系统发生树的同一分支。MhPCS基因为组成型表达,各器官表达水平存在差异,其中根的表达量最高;100μmol.L-1CdSO4处理48 h内,MhPCS基因的表达量迅速上升;不同金属离子对其上调表达的诱导能力为:Cd2+>Cu2+>Pb2+。这为揭示重金属胁迫下湖北海棠环境适应的分子机制奠定了基础。     

番木瓜诱导型基因CpPGIP2启动子的克隆与分析

【目的】获得诱导型基因Cp PGIP2启动子,并分析该启动子的功能。【方法】以番木瓜Cp PGIP2基因c DNA序列(HQ660394)搜索番木瓜基因组DNA序列,获得一段约2 000 bp的5’端上游DNA序列(ABIM01005077),参照该序列设计PCR特异引物,以番木瓜叶片DNA为模板克隆该启动子,并进行生物信息学分析。将3个启动子片段替换p CAMBIA1301载体中的Ca MV 35S启动子,构建缺失启动子表达载体,以GUS瞬时表达检测缺失启动子表达活性。【结果】获得了Cp PGIP2起始密码子上游长为1 981 bp的调控序列,经分析该序列含有真菌、脱落酸、赤霉素、光照等多种响应元件,为诱导型启动子。构建了1个启动子全长表达载体和2个5’端缺失启动子表达载体,分别命名为p D0-1981、p D1-1204和p D2-261。启动子在番木瓜果肉中的瞬时表达显示,长度为1 204 bp的启动子表达活性最强。并且,该启动子在根、茎、叶、果肉和愈伤组织中均有不同程度的表达,在近外果皮的果肉处和根部表达最明显。【结论】本研究获得了Cp PGIP2启动子序列,确定了表达活性最强的启动子长度,初步分析了启动子的表达模式和顺式作用元件,为进一步深入研究Cp PGIP2基因功能以及将该启动子应用于植物基因工程育种奠定了基础。     

香樟两个DREB1转录因子基因的分离及其对不同胁迫的响应分析

以香樟（Cinnamomum camphora）实生苗为试验材料,利用同源克隆结合RACE技术获得了两个冷诱导转录因子基因CBF/DREB1的cDNA全长序列,命名为CcCBFc和CcCBFd,GenBank登录号分别为KP336741和KP336742。序列分析显示这两个基因均没有内含子,cDNA全长为897和1 010 bp,开放阅读框分别为654和621 bp,编码217和206个氨基酸,预测蛋白分子量分别为24.0和22.9 kD,等电点分别为5.26和8.58。基于氨基酸序列的同源性比对和系统进化树分析表明,这两个基因均属于DREB1家族,与双子叶植物进化关系较近。实时定量PCR结果显示,CcCBFc和CcCBFd都能被低温（4 ℃）、干旱（20%PEG）、盐（250 mmol ? L-1 NaCl）和ABA（100 μmol ? L-1）诱导,表明CcCBFc和CcCBFd可能在香樟应对非生物胁迫过程中发挥重要作用。     

Isolation of a citrus ethylene-responsive element binding factor gene and its expression in response to abiotic stress,girdling and shading

Information about citrus ethylene-responsive element binding factor (ERF) genes and their functions in fruit ripening or in stress tolerance is still scarce. In the present study, one of ERF genes, CitERF was isolated from fruit of Citrus unshiu with a maximal putative open reading frame encoding 207 amino acids. The deduced protein contains a region rich in acidic amino acids, an AP2/ERF domain and a KRRK nuclear localization signal. It belongs to group B of Class I in the ERF subfamily in which MdERF2 (Malus × domestica ethylene-response factor 2) and PsERF1b (Prunus salicina ethylene-response factor 1b) were involved in the progress of fruit ripening. CitERF mRNA level in fruit peel and pulp increased obviously along with fruit ripening. However, its expression could be reduced significantly by treatments of total shading and fruit-bear-shoot girdling plus defoliation during fruit ripening. As for the response to abiotic stresses, CitERF expression was found to be induced continuously during the treatment of 10% polyethylene glycol. On the other hand, it could be induced to high level at 1 h after the treatment of 4 °C or 250 mM NaCl and then declined continuously. Taken together, the results suggested that CitERF may play an important role in some biological processes during fruit ripening and in improving tolerance to drought, low temperature and salt stress.     

Molecular cloning and characterisation of a germin-like protein gene in spinach (SoGLP)

Summary

Germin-like proteins (GLPs) are ubiquitous plant glycoproteins that have been implicated in various plant physiological and developmental processes. In this study, a cDNA clone, designated SoGLP, encoding a germin-like protein from spinach (Spinacia oleracia L.) was isolated and characterised. SoGLP encodes a 208-amino-acid polypeptide with a predicted molecular mass of 22.54 kDa and a pI of 5.95. Phylogenetic analysis indicated that SoGLP belonged to sub-family 3 of the GLP family. Sub-cellular localisation of an SoGLP-GFP fusion protein appeared to detect the protein on the cell walls in transgenic tobacco plants. Real-time quantitative RT-PCR analysis showed that the level of expression of SoGLP in the salt-resistant spinach cultivar (‘Chaoji’) was generally higher than in the salt-sensitive spinach cultivar (‘Daye’) grown in 160 mM nitrate ions for 0.5, 3.0, or 6.0 h. Expression of the SoGLP gene was also induced by other abiotic stresses including polyethylene glycol (PEG), NaCl, salicylic acid (SA), or H₂O₂ treatment. Our results indicate that SoGLP could play important roles during high nitrate stress or under other abiotic stresses.     

杨梅酸性转化酶基因cDNA分离及表达分析

杨梅果实富含蔗糖,酸性转化酶是蔗糖代谢关键酶,根据植物酸性转化酶基因保守区序列设计引物,提取杨梅叶片RNA,逆转录获得cDNA,以此为模板通过PCR技术扩增到长度为516bp的基因片段,克隆入pMD18-T载体中,命名为MrIVR1(GenBank:DQ339699)。测序及同源性检索表明,该基因推导氨基酸序列与君子兰、葡萄、草莓、胡萝卜等酸性转化酶基因氨基酸序列同源性为60%～69%。运用ClustalX软件对植物转化酶基因进行了系统树分析,结果显示,MrIVR1编码的蛋白质属于细胞壁酸性转化酶。半定量RT-PCR表达分析显示,MrIVR1基因在杨梅果实发育早期表达量最高,随着果实的发育表达量下降,在成熟果实中表达水平较低。     

干旱胁迫下八棱海棠PCD特征检测及类caspase基因片段的克隆与分析

【目的】为了探明类caspase蛋白酶参与植物PCD的机制,【方法】以八棱海棠(Malus robusta Rehd.)实生苗为试验材料,对植株进行轻度干旱胁迫,检测叶片PCD的发生,并应用RT-PCR技术克隆类caspase基因。【结果】结果表明,轻度干旱胁迫后3周的叶片出现细胞染色质凝聚、胞质皱缩、细胞核变形等细胞凋亡的形态学特征。提取叶片DNA,观察到DNA呈现明显的"DNA Ladder",表现出典型的细胞程序性死亡的生化特征。在此基础上提取叶片RNA,采用RT-PCR获得长度为555 bp的类caspase基因片段。【结论】经同源性分析该片段与其他果树的同源序列相似性超过80%,提示该区段为保守序列。比对苹果基因组,八棱海棠基因组中存在半胱氨酸蛋白酶基因,该基因为单拷贝,并且存在2个同源基因,这为揭示干旱胁迫下八棱海棠调控PCD的机理及环境适应的分子机制奠定了基础。