大豆UBC基因家族鉴定及GmUBC46基因的功能初步分析

泛素/26S蛋白酶体途径在植物响应非生物胁迫反应中起着重要的作用。E2(泛素结合酶,UBC)是蛋白质泛素化中重要的泛素结合酶,与E1和E3共同参与蛋白降解途径。本研究通过构建隐马尔可夫模型,鉴定了54个大豆UBC基因,通过进化树分析将该家族成员分为11个亚家族(A-K)。蛋白保守结构域分析表明,GmUBC家族蛋白成员大部分含有保守Motif 1、Motif 2与Motif 3,且均属于泛素结合酶保守结构域。组织定位分析表明大部分GmUBC家族基因成员在大豆根、茎、叶、花等组织中有所表达。转录组数据表明有20个GmUBC基因在干旱、盐或冷胁迫下具有不同的表达模式,启动子顺式作用元件分析发现其胁迫响应过程可能与激素信号转导相关。进一步通过qRT-PCR发现GmUBC46基因能积极响应干旱、盐或冷胁迫诱导上调表达。通过酵母功能验证表明,GmUBC46基因降低了对干旱或盐胁迫的耐受性。综上,本研究初步阐明了大豆UBC基因家族的基本特性及GmUBC46基因的耐逆功能,为后续研究提供了重要依据和参考价值。     

大黄鱼ubc9基因的克隆和组织表达

类泛素化(sumoylation)是一种重要的转录后修饰过程.激活的SUMO(small ubiquitin-related modifer)和E2结合酶结合稳定后共价结合到底物上,从而完成对底物的类泛素化.UBC9(ubiquitin-conjugating enzyme)作为惟一的E2结合酶,在完成类泛素化中起着重要作用.从已构建的大黄鱼性腺线性化cDNA文库中筛选出ubc9同源片段,用SMART-RACE方法克隆得到了846 bp的全长cDNA序列.该序列编码一个由158个氨基酸组成的蛋白.该蛋白序列与已知的UBC9高度同源,含UBC保守结构域和UBC9激活位点区域.实时定量PCR分析ubc9基因在各组织器官的表达,结果发现,ubc9基因在大黄鱼的性腺中大量表达.推测UBC9在大黄鱼性腺发育中起重要的生物学作用.     

大豆E2泛素结合酶基因GmUBC1的克隆及在拟南芥中的异源表达

E2泛素结合酶(ubiquitin-conjugating enzyme)参与植物的抗逆、生长发育等多种生物途径的调控,其功能研究在拟南芥(Arabidopsis thaliana)中的报道较多,但在重要经济作物大豆(Glycine max)中鲜有报道。本研究从大豆"南农94-16"中克隆了1个与大豆子叶折叠突变体可能相关的基因Glyma.12G161200,序列分析结果表明该基因编码一个E2泛素结合酶,因此将其命名为GmUBC1。该基因编码区全长为462 bp,编码一个含153个氨基酸的蛋白,预测其分子量为17.25 kDa,等电点为6.74。利用qRT-PCR技术对GmUBC1在大豆不同组织中的表达模式及其对不同非生物胁迫和激素处理的响应模式进行分析,发现该基因在开花后40d种子中表达量最高,在PEG、低温、JA和ABA处理下表达下调。亚细胞定位分析发现GmUBC1蛋白在整个细胞内都有表达。进一步在拟南芥中异源表达GmUBC1,发现转基因株系的千粒重和总氨基酸含量显著提高,表明异源过表达GmUBC1可以调控种子重量和氨基酸含量,为大豆品质改良提供了基因资源。     

大黄鱼Lc-UBE2D4基因的克隆及其表达分析

泛素缀合酶E2是蛋白质泛素化系统中的关键酶,对降解的靶蛋白的特异性选择起决定性作用,参与了细胞内80%以上的蛋白降解,对于细胞周期调控、细胞凋亡、基因转录及表达等生理活动具有重要的调控作用。本研究从已构建的大黄鱼(Larimichthys crocea)性腺线性化c DNA文库中筛选出泛素缀合酶(Ubiquitin-conjugating enzymes E2 D4,Lc-UBE2D4)同源基因片段,利用SMART-RACE方法克隆出其全长c DNA序列,并利用荧光定量PCR技术检测其在大黄鱼不同组织和性腺不同发育阶段的差异表达情况。结果显示,Lc-UBE2D4基因全长798 bp,其中ORF为441 bp,可编码147个氨基酸,含有一段典型的泛素缀合酶UBC结构域及其半胱氨酸激活位点。定量PCR结果分析发现,Lc-UBE2D4在脾脏和性腺中表达量较高,在脾脏和精巢的表达水平极显著高于其他各组织(P0.01),同时在卵巢中的表达亦显著高于除脾脏和精巢外的其他各组织(P0.05);通过对Lc-UBE2D4基因在性腺不同发育阶段表达模式的研究发现,该基因在精巢3个时期中的表达水平显著高于各发育阶段的卵巢(P0.05),推测Lc-UBE2D4基因在大黄鱼性腺发育过程中起着重要的调节作用,脾脏中的高表达也暗示其可能参与免疫机能。     

小白菜BcUBCE2基因的克隆及表达分析

采用cDNA-AFLP和RACE技术从小白菜中克隆得到泛素结合酶E2基因(ubiquitin conjugating enzyme E2),命名为BcUBCE2。序列分析表明,BcUBCE2基因cDNA全长830bp,包含1个456bp的开放阅读框,编码152个氨基酸。结构分析发现,该序列包含一个泛素结合酶E2活性位点和一个高度保守的半胱氨酸。进化分析显示,小白菜BcUBCE2蛋白同拟南芥E2蛋白的亲缘关系最近。qRT-PCR分析表明,BcUBCE2基因在小白菜根、茎、叶中均有表达,铜处理10d时BcUBCE2基因的表达量最高。研究认为,BcUBCE2基因可能在铜胁迫响应中发挥重要作用。     

马铃薯泛素结合酶基因StUBC17的克隆与功能分析

泛素结合酶(Ubiquitin-conjugating Enzyme E2,UBC)与植物生长发育和抗病反应密切相关。为了解泛素结合酶基因对植物抗晚疫病的贡献,从马铃薯栽培种"合作88"中克隆出一个E2泛素结合酶基因,命名为StUBC17,并对其受晚疫病菌诱导表达特性及功能进行分析。利用病毒诱导的基因沉默(Virus-induced gene silencing,VIGS)技术降低本氏烟(Nicotiana benthamiana)中StUBC17同源基因(NbUBC)的转录水平,再接种晚疫病菌进行抗病性鉴定。进一步在基因沉默植株上瞬时表达马铃薯抗病基因和其相应的无毒基因(R3a+AVR3a、R3b+AVR3b和Rx+CP)及INF1。StUBC17编码区全长447 bp,编码148个氨基酸,其蛋白分子量为16.52 kD,理论等电点为7.72。表达分析结果表明,StUBC17受晚疫病菌诱导表达。抗病鉴定结果显示,与对照植株相比,NbUBC沉默植株的抗病性显著降低。沉默NbUBC不影响过敏反应(Hypersensitive responses,HR)的发生。StUBC17是植物防御晚疫病所需,但该基因的沉默并不影响R3a、R3b、Rx及INF1介导的HR反应。     

植物泛素结合酶E2功能研究进展

泛素-26S蛋白酶体途径是细胞内蛋白质选择性降解的重要途径,广泛参与植物生长发育相关过程。该途径中关键酶主要包括泛素活化酶(E1)、泛素结合酶(E2)和泛素连接酶(E3),对靶蛋白泛素化起重要作用。在简单概述泛素化过程的基础上,主要对近年来植物E2蛋白在DNA修复、光周期和维管分化调控,缺素及抗逆胁迫响应中的功能进行综述,为今后该蛋白功能的深入研究及木本植物中该功能基因的发掘奠定基础。     

香蕉泛素结合酶基因MaUCE2在非生物胁迫下的表达分析

为了研究泛素结合酶(ubiquitin-conjugating enzyme)E2和植物抗逆性的关系,对香蕉苗进行了非生物胁迫(干旱、低温、盐胁迫)处理,利用荧光定量PCR技术分析了香蕉泛素结合酶基因MaUCE2在不同胁迫条件下的表达特性。结果表明,MaUCE2表达量随着干旱程度的加重而逐步上升,在高度干旱胁迫下MaUCE2的表达量最高;低温胁迫可以诱导MaUCE2的表达,其表达量随着温度的降低而逐步升高,当温度降低到5℃时,MaUCE2的表达量最高;而在盐胁迫下,MaUCE2的表达量与对照相比略有上升,上述结果表明MaUCE2基因表达受非生物胁迫的诱导。     

牵牛胁迫应答基因PnLOG2的克隆及功能验证

RING型E3泛素连接酶在植物应答非生物胁迫过程中发挥着重要功能。该研究从圆叶牵牛中克隆出RING型E3泛素连接酶基因PnLOG2,该基因序列号为XM_019321049.1。利用ORF Finder预测PnLOG2基因编码开放阅读框长度为912 bp (51~992 bp),编码313个氨基酸,蛋白分子质量34.38 kD,理论等电点为5.14。系统发育分析表明,PnLOG2基因与番茄亲缘关系最近。组织特异性分析表明,PnLOG2基因在牵牛不同组织均有表达,在老茎和新叶中表达量较高。qRT PCR分析结果表明,PnLOG2基因在圆叶牵牛根和叶中受干旱、盐碱胁迫诱导显著上调表达。通过异源表达PnLOG2基因于酵母细胞中,发现干旱、盐碱胁迫下PnLOG2基因提高了重组酵母的耐盐和耐旱能力,但降低了对碱的耐受性。该研究初步阐明了PnLOG2基因在干旱、盐碱胁迫下的功能,为进一步研究RING型E3泛素连接酶在非生物胁迫中的机理提供了理论依据。     

橡胶草TkAPC10基因的鉴定及其表达模式分析

APC/C是一类泛素连接酶E3复合体,在调控细胞周期过程中发挥重要作用。为了揭示橡胶草APC/C蛋白复合体的功能,鉴定了橡胶草TkAPC10基因,并对其表达模式进行了分析,初步确定了其功能。TkAPC10基因的ORF为579 bp,编码192个氨基酸,其基因组DNA序列为1 092 bp,包含6个外显子和5个内含子。基因组分析发现,TkAPC10以单拷贝的形式存在,其启动子序列除了含有TATA-box和CAAT-box增强子元件外,还有ABA、JA、光以及逆境响应相关的顺式作用元件。系统进化关系分析发现,不同物种的APC10蛋白具有很高的同源性,TKAPC10与莴苣LsAPC10的相似性最高达到99%,而与其他菊科植物的APC10蛋白相似性也达到95%以上。进一步采用qRT-PCR技术对TKAPC10的表达模式进行分析,结果表明,该基因在细胞分裂旺盛的组织（花、叶和根）中的表达量显著高于细胞分裂活动相对缓慢的组织（花梗）。外源ABA处理后,TKAPC10基因转录水平显著下降;而MeJA和ET处理后,该基因显著上调表达。经PEG6000以及甘露醇处理后,TKAPC10表达水平显著下降;而高盐胁迫显著诱导该基因的表达。TKAPC10基因参与橡胶草细胞分裂、激素信号以及非生物胁迫响应过程的调控。     

A miRNA involved in phosphate-starvation response in Arabidopsis

Although microRNAs (miRNAs) have been documented to regulate development in plants and animals , the function of miRNAs in physiology is unclear. miR399 has multiple target sites in the 5' untranslated region (UTR) of a gene encoding a putative ubiquitin-conjugating enzyme (UBC) in Arabidopsis thaliana. We report here that miR399 was highly induced, whereas the target UBC mRNA was reduced by low-phosphate (Pi) stress. In transgenic plants with constitutive expression of miR399, UBC mRNA accumulation was suppressed even under high Pi. The expression of transgene UBC mRNA with 5' UTR miR399 target sites, but not the one without 5' UTR, was reduced under low-Pi condition. Furthermore, transgenic Arabidopsis plants with constitutive expression of miR399 accumulated more Pi than the wild-type, and transgenic plants expressing the UBC mRNA without 5' UTR (miRNA-deregulated) showed less inhibition of primary root growth and less induction of a Pi transporter gene by low-Pi stress than those of wild-type plants. We conclude that miR399 downregulates UBC mRNA accumulation by targeting the 5' UTR, and this regulation is important for plant responses to Pi starvation. The results suggest that miRNAs have functional roles for plants to cope with fluctuations in mineral-nutrient availability in the soil.     

Evaluation of reference genes for real-time quantitative PCR studies in

ABSTRACT: BACKGROUND: The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata. RESULTS: The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1a, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-[increment][increment]CT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-[increment][increment]CT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments. CONCLUSIONS: We recommend the use of RDN5.8, UBC13, and PGK1 alone or the combination of RDN5.8 plus UBC13 or PGK1 as reference genes for RT-qPCR analysis of gene expression in C. glabrata following azole treatment. In contrast, we show that ACT1 and other commonly used reference genes (GAPDH, PPIA, RPL13A, TUB1, etc.) were not validated as good internal controls in the current model.     

A comparative proteomic analysis for capsaicin-induced apoptosis between human hepatocarcinoma (HepG2) and human neuroblastoma (SK-N-SH) cells

The endogenous ROS levels were increased during HepG2 apoptosis, whereas they were decreased during SK-N-SH apoptosis in response to capsaicin treatments. We used 2-DE-based proteomics to analyze the altered protein levels in both cells, with special attention on oxidative stress proteins before and after capsaicin treatments. The 2-DE analysis demonstrated that 23 proteins were increased and 26 proteins were decreased significantly (fold change>1.4) in capsaicin-treated apoptotic HepG2 and SK-N-SH cells, respectively. The distinct effect of capsaicin-induced apoptosis on the expression pattern of HepG2 proteins includes the downregulation of some antioxidant enzymes including aldose reductase (AR), catalase, enolase 1, peroxiredoxin 1, but upregulation of peroxiredoxin 6, cytochrome c oxidase, and SOD2. In contrast, most antioxidant enzymes were increased in SK-N-SH cells in response to capsaicin, where catalase might play a pivotal role in maintenance of low ROS levels in the course of apoptosis. The global gene expression for oxidative stress and antioxidant defense genes revealed that 84 gene expressions were not significantly different in HepG2 cells between control and capsaicin-treated cells. In contrast, a number of oxidative genes were downregulated in SK-N-SH cells, supporting the evidence of low ROS environment in apoptotic SK-N-SH cells after capsaicin treatment. It was concluded that the different relationship between endogenous ROS levels and apoptosis of two cancer cells presumably resulted from complicated expression patterns of many oxidative stress and antioxidant genes, rather than the individual role of some classical antioxidant enzymes such as SOD and catalase.     

胡杨PeNAC121基因启动子的分离鉴定和胁迫应答模式分析

为了探究NAC转录因子家族成员在胡杨（Populus euphratica）逆境胁迫中的响应和调控机制,利用PCR技术从胡杨中克隆了PeNAC121基因的启动子序列,并采用生物信息学工具对该启动子的结构特征进行了分析,最后利用该启动子驱动GUS报告基因在三倍体毛白杨（Populus tomentosa）中表达,并对获得的转基因植株采用不同胁迫处理后进行了GUS染色和酶活性定量分析。结果表明,克隆获得的PeNAC121基因的启动子长度为1 997 bp（起始密码子ATG上游）,启动序列中除了含有大量的光响应元件,还含有多个与非生物逆境胁迫和激素响应相关的元件,如低温响应元件LTR、干旱响应元件MBS、防卫和胁迫响应元件TC-rich repeats、脱落酸（ABA）响应元件、以及赤霉素（GA）响应元件等。基因的组织表达模式检测结果显示,PeNAC121基因主要在茎中表达,在根和叶中的表达较少。GUS组织化学染色和酶活性检测结果表明,胡杨PeNAC121启动子显著受到NaCl、甘露醇、ABA和4 ℃低温的诱导表达。由上述结果推测PeNAC121基因与胡杨的逆境胁迫应答密切相关,表明该基因的启动子是一个能够应答多种逆境胁迫的诱导型启动子。本研究为阐明PeNAC121基因在胡杨逆境响应和调控中的作用机制提供理论参考。     

Repression of the floral transition via histone H2B monoubiquitination

The Rad6-Bre1 complex monoubiquitinates histone H2B in target gene chromatin, and plays an important role in positively regulating gene expression in yeast. Here, we show that the Arabidopsis relatives of the yeast Rad6, ubiquitin-conjugating enzyme 1 (UBC1) and UBC2, redundantly mediate histone H2B monoubiquitination, and upregulate the expression of FLOWERING LOCUS C ( FLC ; a central flowering repressor in Arabidopsis) and FLC relatives, and also redundantly repress flowering, the developmental transition from a vegetative to a reproductive phase that is critical in the plant life cycle. Moreover, we have found that Arabidopsis relatives of the yeast Bre1, including HISTONE MONOUBIQUITINATION 1 (HUB1) and HUB2, also upregulate the expression of FLC and FLC relatives, and that HUB1 genetically interacts with UBC1 and UBC2 to repress the floral transition. These findings are consistent with a model in which HUB1 and HUB2 specifically interact with and direct UBC1 and UBC2 to monoubiquitinate H2B in developmental genes, and thus regulate developmental processes in plants.     

Stress resistance in Saccharomyces cerevisiae is strongly correlated with assembly of a novel type of multiubiquitin chain.

The covalent attachment of ubiquitin (Ub) to short-lived or damaged proteins is believed to be the signal that initiates their selective degradation. In several cases, it has been shown that the proteolytic signal takes the form of a multi-Ub chain in which successive Ub molecules are linked tandemly at lysine 48 (K-48). Here we show that Ub molecules can be linked together in vivo at two other lysine positions, lysine 29 (K-29) and lysine 63 (K-63). The formation of these alternative linkages is strongly dependent on the presence of the stress-related Ub conjugating enzymes UBC4 and UBC5. Furthermore, expression of Ub carrying a K-63 to arginine 63 substitution in a strain of Saccharomyces cerevisiae that is missing the poly-Ub gene, UBI4, fails to compensate for the stress defects associated with these cells. Taken together, these results suggest that the formation of multi-Ub chains involving K-63 linkages plays an important role in the yeast stress response. In broader terms, these results also suggest that Ub is a versatile signal in which different Ub chain configurations are used for different functions.