菜豆多聚泛肽基因在重金属胁迫下的表达

差别筛选HgCl2胁迫的菜豆(PhaseolusvulgarisL.)幼苗叶片cDNA库,分离出两个重金属胁迫相应基因PvSR5和PvSR51(Phaseolusvulgarisstress_relatedgene)片段。cDNA和氨基酸序列分析表明PvSR5和PvSR51分别编码一种多聚泛肽。Northernblot分析表明多聚泛肽是组成性表达蛋白,主要在根中表达,叶片和茎中表达较少;Hg、Cd、Cu和Zn等重金属、高温和水杨酸能强烈地刺激其在叶片中的表达,而受伤几乎没有影响。推测多聚泛肽在抵抗重金属胁迫和提高植物的抗逆性方面有重要作用。     

菜豆重金属胁迫响应基因:cDNA克隆及其表达分析

通过差别筛选HgCl2胁迫下的菜豆叶片cDNA库,分离出7组不同的cDNA克隆（Phaseolusvulgarisstress-relatedprotein,PvSR1～7）。cDNA序列和同源性分析结果表明：PvSR1编码富含脯氨酸细胞壁蛋白（PRP）,PvSR2和PvSR7编码新的HgCl2胁迫相关蛋白,PvSR3编码脱水蛋白（dehydrin）,PvSR4编码病原相关（PR）蛋白,PvSB5编码polyubiqui－tin,PvSR6编码DuaJ－like蛋白。HgCl2胁迫可强烈地请导PvSR2和PR蛋白基因的表达,并能提高PRP,、dehydrinlike和polyubiq－uitin基因的转录水平。这些蛋白质共同作用可能对维持细胞的正常代谢和抵抗重金属胁迫方面有重要作用。     

菜豆病程相关蛋白基因在重金属胁迫下的表达分析

为探讨植物抗重金属的分子机理 ,差别筛选了 Hg Cl2 胁迫的菜豆 ( Phaseolus vulgaris L.)叶片 c DNA库 ,分离出一个重金属胁迫响应基因 Pv SR4克隆 .c DNA和氨基酸序列分析表明 Pv SR4编码一种细胞内病程相关蛋白 ,该蛋白具有 RNase活性 .Northern blot分析表明 Pv SR4基因在正常生长条件下的叶片中不表达 ,重金属 ( Hg、Cd、As、Zn和 Cu等 )和水杨酸能强烈地诱导其基因的表达 ,受伤也能促进该基因的转录 ,而热胁迫几乎没有调节作用 .推测 Pv SR4蛋白在诱导植物的抗逆性和抵抗重金属胁迫方面有重要作用     

DnaJ—like基因在不同环境胁迫下的表达研究

ＰｖＳＲ６（Ｐｈａｓｅｏｌｕｓ ｖｕｌｇａｒｉｓ ｓｔｒｅｓｓ－ｒｅｌａｔｅｄ）基因编码一种菜豆ＤｎａＪ－ｌｉｋｅ蛋白。Ｎｏｒｔｈ－ｅｒｎ ｂｌｏｔ结果表明：ＰｖＳＲ６基因在未处理的菜豆叶片中表达较少,重金属（Ｈｇ＾２＋和Ｃｄ＾２＋）、机械损伤、ＵＶ、高温和水杨酸等环境胁迫能强烈地促进其基因的转录,推测ＤｎａＪ－ｌｉｋｅ蛋白在保护细胞膜和酶蛋白的结构和功能及提高植物的抗逆性方面有重要作用。     

菜豆重金属响应基因PvSR2在烟草中的表达及镉抗性分析

重金属特异诱导基因PvSR2(Phaseolus vulgaris stress-related)是从法国菜豆中克隆出来的, 为了研究该蛋白能否提高植物的抗重金属能力, 将PvSR2基因插入到植物转化中间载体pCAMBIA2301中CaMV 35S启动子的下游, 用根癌农杆菌介导的叶盘法将其导入烟草中, 在含有100 mg/L Kan的MS培养基上筛选, 获得了转基因植株. PCR和Southern杂交结果表明PvSR2已整合在烟草基因组中, GUS和Northern分析表明PvSR2在转基因烟草中获得表达. 重金属抗性实验表明: 与野生型烟草相比, PvSR2转基因烟草具有较高的抗重金属镉(Cd)的能力. 组织Cd含量分析显示: 在低浓度Cd (0.5~0.75 mmol/L)处理时, Cd在PvSR2转基因烟草与野生型烟草根中的累积量没有明显的差别, 而在高浓度Cd(0.1 mmol/L)胁迫下, 转基因烟草根中Cd的累积量低于野生型烟草, 说明PvSR2的表达能够提高植物的抗重金属能力, 同时表明PvSR2可能与重金属在植物中的运输和积累有一定关系.     

表达序列标签数据库搜索鉴定小鼠UBAP1基因及其数字化表达分析

UBAP1(ubiquitin associated protein 1)基因是最近克隆的一个定位于人类染色体9p21-22鼻咽癌杂合性丢失高频区的泛肽相关蛋白家族新成员.为了深入研究UBAP1基因的功能,利用计算机对表达序列标签(expressed sequence tag, EST)、UniGene等数据库进行综合搜索分析,结合cDNA克隆测序的方法, 成功地获得了UBAP1基因在小鼠中的同源基因.小鼠UBAP1基因cDNA全长为2 676 bp,编码一个由441个氨基酸组成的蛋白质,在其蛋白质C端只有一个泛肽相关功能域（UBA domain）.与人UBAP1基因相比,两者编码的氨基酸序列有89%相同.基于EST的数字化表达分析显示UBAP1基因在小鼠正常组织中广泛高表达.     

沙冬青AmNAC3转录因子基因在抗旱性和抗寒性中的功能鉴定

为了研究强抗逆植物沙冬青Am NAC3转录因子基因在抗旱性和抗寒性中的功能,首先利用半定量RT-PCR方法对该基因进行了表达分析。结果表明,在室内培养的沙冬青幼苗中,Am NAC3有一定量的基础表达,在干旱胁迫下其转录水平明显上调,而在低温胁迫下其表达上调较弱。然后利用5'RACE技术获得该基因的5'端序列及全长cDNA序列,并利用RT-PCR方法克隆到其全长编码区(846bp)。将编码区片段构建到植物表达载体上,利用农杆菌介导法获得转基因拟南芥。进一步分析表明,转基因拟南芥对于干旱和低温胁迫的抗性表型与野生型无明显差异,但其离体叶片的失水率和气孔开度均大于野生型。此外,转基因幼苗中气孔开闭相关基因ABI1和ABI2的表达量降低。这些结果表明,Am NAC3可能主要在响应干旱胁迫和调节气孔开闭及叶片保水性中发挥功能,而在抵抗低温胁迫中无明显作用。     

茶树冷诱导基因RAV的克隆与表达特性分析

对利用cDNA-AFLP技术所获得的茶树低温诱导差异表达片段TDF,通过RACE方法获得含完整编码区序列的茶树RAV基因cDNA克隆,其开放阅读框编码361个氨基酸,包含两个保守的结构域AP2和B3,与多种植物RAV蛋白具有高度同源性。qRT-PCR分析表明,茶树RAV基因受低温、乙烯、NaCl等上调表达,最大表达量分别是诱导前的5.8、10.0和1.9倍。在成熟叶片、芽、嫩茎中RAV基因表达量相近,花蕾和嫩根中表达较低,而在种子中不表达。推测该基因在组织中的表达受到严格控制以及在响应非生物胁迫中发挥重要作用。     

茶树CsNCED2基因的克隆和表达分析

该研究以铁观音茶树品种叶片为材料,通过RT-PCR技术,克隆了茶树脱落酸(ABA)合成途径关键限速酶——9-顺式环氧类胡萝卜素裂解双加氧酶(9-cis-epoxycarotenoid dioxygenase,NCED)基因的全长cDNA序列。该基因cDNA全长1 931bp,包含1 821bp完整开放阅读框,共编码606个氨基酸残基。NCBI同源分析结果表明,与葡萄VvNCED2相似性最高(78%),命名为CsNCED2(NCBI登录号:MF765770)。氨基酸序列分析显示,其具有NCED家族的FLNO2258保守结构域,以及MIAHPKxDP和HDFAITE保守结构域序列;在保守区存在4个Fe2+活性组氨酸结合位点,N-端含有叶绿体转运肽。实时荧光定量PCR分析表明,CsNCED2基因在铁观音叶、茎和花中表达量较高;白茶萎凋和乌龙茶做青均可以诱导CsNCED2基因显著上调表达;除干旱胁迫抑制CsNCED2表达外,ABA和低温胁迫均能够诱导CsNCED2基因显著上调表达。表明CsNCED2基因在茶树ABA合成代谢以及胁迫响应中发挥重要作用。     

与泛肽途径可能相关的新基因UBAP1的克隆和表达分析

在先前确定了鼻咽癌9p21-22区域的一个最小共同缺失区内的基础上,为了筛选和克隆鼻咽癌相关的修选抑瘤基因,应用EST介导的定位候选克隆策略,用RT－PCR及Northern杂交检测了22个表达序列标签(expressed sequence tag,EST）在鼻咽癌细胞株HNE－1和原代培养的正常鼻咽上皮细胞中的表达水平,发现其中一个EST w56112在鼻咽癌细胞株HNE－1中的表达显著下调,RNA印迹显示其代表一转录本为2.7kb的基因。进一步运用cDNA测序和RACE方法克隆了该EST代表的基因全长cDNA,Genbank登录呈AF222043,同时结合生物信息学方法克了该基因在小鼠中的同源基因,Genbank登录AF275549。该基因cDNA全长2.7kb,编码由502个氨基酸组成的、分子质量为55kD的蛋白质。数据库分析显示该基因编码的蛋白质羧基段含有两个重要的泛肽相关结构域（UBA domain）,属于泛肽相关蛋白家族的一个新成员,因此征得国际人类基因组命名委员会同意,将其命名为UBAP1基因。运用Northern杂交和 RT-PCR方法检测发现UBAP1基因在所检测的人和小鼠的组织中广泛表达。采用RT－PCR和直接测序的方法,未能发现UBAP1基因编码区在鼻咽癌细胞株HNE－1和10例鼻咽癌活检标本中存在突变。UBAP1基因作为一个泛肽相关蛋白家族的新成员,有可能参与泛肽信号途径;结合其在9p的定位信息及在鼻咽癌中的表达下调。有等对UBAP1基因进行为精细的突变分析,以进一步研究其表达下调参与鼻咽癌发生发展的可能机制。     

Cloning and expression analysis of SKn-type dehydrin gene from bean in response to heavy metals

A heavy metal responsive gene PvSR3 (GenBank accession number U54703) encoding an acid dehydrin was isolated from a mercuric chloride-treated bean (Phaseolus vulgaris L.) leaf cDNA library by differential screening using cDNAs derived from treated and untreated plants. The PvSR3 cDNA is 981-bp long and has a 606-bp open-reading frame with a 202-residue-deduced amino acid sequence. The PvSR3 sequence contains two conserved repeats of the characteristic lysine-rich K segment (EKKGIMDKIKEKLPG) preceded by an 8-serine residue stretch, whereas the Y segment (DEYGNP) conserved motif is absent. The deduced protein has a calculated molecular weight of 23 kDa and an isoelectric point of 5.2. Sequence similarity and comparative analysis showed that PvSR3 shares 70 and 73% similarity with the dehydrin of poplar and pepper, respectively. Southern hybridizations indicated that PvSR3 was a low copy-number gene. Northern blot analysis revealed that PvSR3 mRNA was weakly detected in seedling leaves. However, the gene expression was strongly stimulated by heavy metals, such as mercury, cadmium, arsenic, and copper, whereas virus infection and salt had little effect on it. In contrast, PvSR3 was not responsive to drought or abscisic acid (ABA), and was downregulated by UV radiation. Furthermore, PvSR3 was upregulated by the exogenous signaling molecules, including salicylic acid (SA) and hydrogen peroxide (H2O2). It is suggested that PvSR3 is extremely related to heavy metal stress, and might play an important role in metal detoxification and resistance to the damage caused by heavy metals.     

Cloning and expression analysis of SKn-type dehydrin gene from bean in response to heavy metals

A heavy metal responsive gene PvSR3 (GenBank accession number U54703) encoding an acid dehydrin was isolated from a mercuric chloride-treated bean (Phaseolus vulgaris L.) leaf cDNA library by differential screening using cDNAs derived from treated and untreated plants. The PvSR3 cDNA is 981-bp long and has a 606-bp open-reading frame with a 202-residue-deduced amino acid sequence. The PvSR3 sequence contains two conserved repeats of the characteristic lysine-rich K segment (EKKGIMDKIKEKLPG) preceded by an 8-serine residue stretch, whereas the Y segment (DEYGNP) conserved motif is absent. The deduced protein has a calculated molecular weight of 23 kDa and an isoelectric point of 5.2. Sequence similarity and comparative analysis showed that PvSR3 shares 70 and 73% similarity with the dehydrin of poplar and pepper, respectively. Southern hybridizations indicated that PvSR3 was a low copy-number gene. Northern blot analysis revealed that PvSR3 mRNA was weakly detected in seedling leaves. However, the gene expression was strongly stimulated by heavy metals, such as mercury, cadmium, arsenic, and coppper, whereas virus infection and salt had little effect on it. In contrast, PvSR3 was not responsive to drought or abscisic acid (ABA), and was downregulated by UV radiation. Furthermore, PvSR3 was upregulated by the exogenous signaling molecules, including salicylic acid (SA) and hydrogen peroxide (H₂O₂). It is suggested that PvSR3 is extremely related to heavy metal stress, and might play an important role in metal detoxification and resistance to the damage caused by heavy metals.     

Cadmium resistance in transgenic tobacco plants enhanced by expressing bean heavy metal-responsive gene <Emphasis Type="Italic">PvSR2</Emphasis>

PvSR2 (Phaseolus vulgaris stress-related gene) has been cloned from French bean and shown to be expressed specifically upon heavy metal treatment. In order to investigate the role of PvSR2 in plant, PvSR2 gene under the control of cauliflower mosaic virus 35S promoter was introduced into tobacco mediated with Agrobacterium tumefaciens LBA4404. The regenerated plantlets were selected on medium with 100 mg/L kanamycin. PCR and Southern blot analysis showed PvSR2 gene was integrated in tobacco genome. Gus and Northern blot analysis indicated PvSR2 gene was expressed in transgenic seedling. The heavy metal resistance assay showed that the transgenic tobacco seedlings with the PvSR2 coding sequence exhibited higher tolerance to Cd compared with wild-type (WT) under Cd exposure. The Cd content accumulated in root between transgenic and WT seedlings had no obvious difference at lower Cd external concentration (0.05-0.075 mmol/L CdCl2), whereas transgenic plant showed a lower root Cd content than the control at higher external Cd concentration (0.1 mmol/L CdCl2). These results suggested that the expression of PvSR2 can enhance the Cd tolerance, and PvSR2 may be involved in Cd transportation and accumulation at the test concentration of 0.1 mmol/L Cd.     

Cadmium resistance in transgenic tobacco plants enhanced by expressing bean heavy metal-responsive gene PvSR2

Heavy metal pollution such as Cd, Hg, Pb, As and Se is an increasing environment problem worldwide. These metals and metalloids have toxic effect on both plants and animals, which are strongly poisonous to metal-sensitive enzymes, resulting in growth inhibition and death of the organism[1]. Contamination of soils with heavy metals, either by natural causes or due to pollution, often has pronounced effects on the vegetation, resulting in the appearance of metallophytes, and heavy-metal tolera…