小麦中小GTP结合蛋白基因TaRop2克隆及其表达分析

小GTP结合蛋白属于Rho家族,是植物特有的一类蛋白,在调控植物生长发育、抗逆和抗病过程中发挥着重要的作用。本研究通过筛选非亲和条锈菌小种CYR32侵染诱导的抗条锈病基因Yr5近等基因系(Taichung29*6/Yr5)c DNA文库,分离获得1个Rop家族基因的全长c DNA序列,命名为TaRop2(Triticum aestivum Rop2)。TaRop2包含1个591 bp的开放阅读框,预测编码含197个氨基酸残基的蛋白质,分子量为21.52 k D,理论等电点为9.49。通过在烟草表皮细胞瞬时表达,发现TaRop2分布于细胞核内和细胞膜上。RT-PCR分析结果表明,在高盐处理、非亲和条锈菌小种CYR32和亲和混合白粉菌菌株侵染时,TaRop2基因的表达水平升高,但被干旱、高温、低温和ABA处理抑制。说明TaRop2可能参与小麦防卫和抗逆反应过程。     

盐胁迫对拟南芥AtPUB18基因的诱导表达及其启动子分析

用300mmol/L NaCl处理拟南芥幼苗,分别于处理后0、1、2、4、8、16、24、48h通过Northern Blot检测其AtPUB18基因的表达量。结果显示:拟南芥AtPUB18基因的表达量受高盐胁迫的诱导而升高,于处理后4h表达量达到最高,处理后16h表达量最低。采用PCR技术克隆AtPUB18的启动子,序列为1 974bp;序列分析发现启动子内含有大量与非生物胁迫相关的顺式作用元件,如HSE、LTR、MBS及ABRE;将启动子克隆到表达载体pCambia1300-221-GUS中,驱动报告基因GUS表达。组织化学染色结果表明,未经过高盐处理的幼苗中GUS基因表达水平很低;300mmol/L NaCl处理后GUS基因表达量显著升高。研究表明,AtPUB18的表达受高盐胁迫诱导,且AtPUB18基因的启动子是一个盐胁迫诱导型启动子。     

水稻rab5B基因在大肠杆菌中的表达、纯化和GTP结合分析

Rab5B类蛋白因为其编码产物的N端具有特殊结构而被认为是一类特殊的蛋白质.水稻rab5B基因Osrab5B是这类蛋白质基因在单子叶植物中的首例发现.将Osrab5B基因的编码序列按正确读码框重组到具有谷胱甘肽硫转移酶(glutathione S-transferase, GST)融合标签的pGEX-4T1表达载体中,转化大肠杆菌,获得了稳定表达目标融合蛋白的菌株,经GSTrapTM柱纯化,获得了纯化的目标融合蛋白.GTP结合试验表明,在原核细胞中表达出的GST-OsRab5B融合蛋白具有体外结合GTP的能力.     

水稻rab5B基因在大肠杆菌中的表达、纯化和GTP结合分析

Rab5B类蛋白因为其编码产物的N端具有特殊结构而被认为是一类特殊的蛋白质.水稻rab5B基因Osrab5B是这类蛋白质基因在单子叶植物中的首例发现.将Osrab5B基因的编码序列按正确读码框重组到具有谷胱甘肽硫转移酶（glutathione S-transferase, GST）融合标签的pGEX-4T1表达载体中,转化大肠杆菌,获得了稳定表达目标融合蛋白的菌株,经GSTrap^TM柱纯化,获得了纯化的目标融合蛋白.GTP结合试验表明,在原核细胞中表达出的GST-OsRab5B融合蛋白具有体外结合GTP的能力.     

花生ASR基因家族特性及其对干旱和盐胁迫的响应

【目的】探究花生ASR基因家族特性及在干旱和盐胁迫响应中的作用,为花生抗旱抗盐新品种的培育提供潜在的基因位点。【方法】通过生物信息学方法对花生ASR家族进行全基因组水平鉴定以及基本特性分析,并借助转录组数据分析其在200 mmol/L NaCl及模拟干旱PEG处理下的表达变化。【结果】（1）通过分析花生栽培种狮头企参考基因组,鉴定到7个花生ASR基因,pI为5.34~6.98,蛋白脂肪系数为23.77~56.84,GRAVY值均为负值,表明这7个蛋白均是亲水性蛋白;（2）AhASR3与AhASR7基因表达模式相似,转录水平较高,基因结构及蛋白结构域和保守基序的位置和数量较相似,motif 5、6、9仅存在于AhASR3与AhASR7蛋白中;（3）AhASR1、AhASR5及AhASR2的启动子区域有干旱诱导MYB转录因子的结合位点,AhASR1、AhASR2及AhASR4的启动子区发现有ABA响应元件;（4）花生盐胁迫处理转录组分析结果显示AhASR2、AhASR3及AhASR7在200 mmol/L NaCl处理后根部出现较明显转录上调;（5）模拟干旱PEG处理转录组数据分析结果显示AhASR1、AhASR3、AhASR4及AhASR7在PEG处理4 h和8 h后,转录水平出现2倍以上上调。【结论】明确花生ASR家族基因和蛋白的基本特性,并鉴定可能参与盐胁迫和干旱胁迫响应的ASR基因,为进一步培育耐盐耐旱花生品种提供重要的目标基因。     

白桦BpZFP4基因启动子克隆和逆境响应元件功能分析

前期研究发现白桦锌指蛋白BpZFP4基因响应多种非生物逆境胁迫。为了深入研究其调控机制,本研究利用染色体步移技术克隆了该基因上游1 360 bp的启动子区域,序列分析结果表明该区域含有多个逆境响应顺式作用元件、激素响应元件、光响应以及病原菌和损伤响应元件等。在此基础上,构建了BpZFP4启动子5'-端一系列缺失片段融合GUS报告基因的表达载体。通过对系列缺失突变体在正常条件以及渗透胁迫、盐胁迫和脱落酸（ABA）处理条件下的GUS基因表达分析,鉴定得到BpZFP4启动子对干旱、盐和ABA应答响应的主要调节区域及可能的顺式作用元件。本研究结果为进一步探究BpZFP4基因应答非生物胁迫和信号分子刺激的调控机理提供了重要的理论依据。     

植物非生物胁迫诱导启动子顺式作用元件的研究方法

非生物胁迫严重影响植物生长发育,降低作物产量。植物通过各种途径忍受或抵抗非生物胁迫,主要表现是各种抗非生物胁迫基因的表达。基因表达受其上游启动子及转录因子的调控,目前对抗非生物胁迫诱导启动子顺式作用元件及转录因子的研究成为热点。本文综述了植物非生物胁迫诱导启动子顺式作用元件及转录因子的研究方法,并展望了顺式作用元件及转录因子研究的方向及前景。     

花生ARAhPR10基因启动子序列的克隆及分析

PR10(pathogenesis-related class10protein)类蛋白与植物的抵御外来病害及系统获得性抗性(SAR)有着紧密联系,本文采用基于PCR的基因组DNA步移法,从抗黄曲霉花生品种粤油20中克隆ARAhPR10(Aspergillus flavus-resistant AhPR10)基因起始密码子ATG上游256bp类似启动子序列,并对其进行植物顺式作用元件数据库PLACE预测分析。结果表明,该类似启动子序列含有4处TATA box和2处CAAT box保守的启动子结构元件,还有6处W-box、1处BIHD1和3处GT-1motif抗逆应答元件,其中W-box常见于PR蛋白的启动子区内参与病程应答。我们初步认为本研究克隆的序列可能是ARAhPR10基因的启动子。     

抗逆基因AtRPK1启动子关键顺式作用元件的研究

为确定拟南芥抗逆相关基因AtRPK1启动子的顺式功能元件,对其启动子区进行了分段克隆。通过5'端缺失方法得到203、316、604、809 bp 4个启动子片段,分别构建成p1300-pro-GUS表达载体,并转入拟南芥,进行GUS染色和GUS定量检测。通过对809 bp全长启动子转基因拟南芥GUS染色发现,转基因拟南芥的叶片、茎、花、根中均有表达,在分生能力强的组织和维管束集中的组织,AtRPK1基因启动子具有较高启动表达能力。5'端缺失启动子检测结果表明,转录起始点到启动子上游-114位点区域包含AtRPK1基因启动子的关键顺式作用元件。对启动子缺失片段转基因植株利用200 mmol·L-1NaCl胁迫3 h后,β-葡萄糖苷酸酶活力定量检测结果表明,在启动子上游-19位点处的GT-1顺式作用元件GAAAAA可能直接与盐胁迫应答相关。     

ACC氧化酶基因AhACOs对花生耐盐性的影响

ACC氧化酶(ACC oxidase,ACO)是催化乙烯合成的关键酶之一,乙烯参与植物的盐胁迫反应过程,而盐胁迫严重影响花生产量。本研究通过对AhACOs基因的克隆及功能验证,探究AhACOs在花生盐胁迫响应中的生物学功能,为花生耐盐品种的选育提供基因资源。以花生耐盐突变体M29的cDNA为模板扩增得到基因AhACO1和AhACO2,与植物表达载体pCAMBIA super1300重组后,通过农杆菌介导的花粉管注射法将重组质粒转化到花育22号中。收获后切取籽仁远胚端部分子叶,利用PCR检测筛选阳性籽仁。利用qRT-PCR分析AhACOs基因表达量,通过毛细管柱气相色谱法检测植株的乙烯释放量。阳性籽仁和对照籽仁种植21 d后浇盐水,观察其表型变化。结果发现,盐胁迫后,转基因植株生长状况好于对照组花育22号,并且其叶绿素相对含量SPAD(soil and plant analyzer development)值和净光合速率(net photosynthesis rate,Pn)均高于对照组花生。另外,AhACO1和AhACO_(2)转基因植株的乙烯释放量分别为对照组花生的2.79倍和1.87倍。这些结果表明AhACO1和AhACO2可显著提高花生的耐盐能力。     

Small GTP binding proteins: Rab GTPases from the brain of Bombyx mori

From a mRNA of the brain of Bombyx mori, we isolated 8 cDNA clones (BRabs), each of which encodes a different member of Rab-protein family. Four of them have more than 80% amino acid identity to the corresponding members of Drosophila Rab proteins. The other 4 proteins show low sequence similarity to any of the known Rab proteins. However, all of them contain the region conserved in rab protein. Using RACE (Rapid Amplification of cDNA ends), the one full-length cDNA clone (BRab14) was isolated. The clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. After purification, the fusion protein was cut with protease to remove GST-Tag and applied to a glutathione S-Sepharose column. The protein bound [(3)H]-GDP with association constant of 1.02 x 10(11) M(-1). Further, the protein was phosphorylated by protein kinase. This result suggests that Rab protein in the brain of Bombyx mori binds GDP or GTP and its function is regulated by phosphorylation.     

Ligand binding to the inhibitory and stimulatory GTP cyclohydrolase I/GTP cyclohydrolase I feedback regulatory protein complexes

GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4), which is an essential cofactor for key enzymes producing catecholamines, serotonin, and nitric oxide as well as phenylalanine hydroxylase. GFRP also mediates feed-forward stimulation of GTP cyclohydrolase I activity by phenylalanine at subsaturating GTP levels. These ligands, BH4 and phenylalanine, induce complex formation between one molecule of GTP cyclohydrolase I and two molecules of GFRP. Here, we report the analysis of ligand binding using the gel filtration method of Hummel and Dreyer. BH4 binds to the GTP cyclohydrolase I/GFRP complex with a Kd of 4 microM, and phenylalanine binds to the protein complex with a Kd of 94 microM. The binding of BH4 is enhanced by dGTP. The binding stoichiometrics of BH4 and phenylalanine were estimated to be 10 molecules of each per protein complex, in other words, one molecule per subunit of protein, because GTP cyclohydrolase I is a decamer and GFRP is a pentamer. These findings were corroborated by data from equilibrium dialysis experiments. Regarding ligand binding to free proteins, BH4 binds weakly to GTP cyclohydrolase I but not to GFRP, and phenylalanine binds weakly to GFRP but not to GTP cyclohydrolase I. These results suggest that the overall structure of the protein complex contributes to binding of BH4 and phenylalanine but also that each binding site of BH4 and phenylalanine may be primarily composed of residues of GTP cyclohydrolase I and GFRP, respectively.     

The Rab GTPase RabG3b functions in autophagy and contributes to tracheary element differentiation in Arabidopsis

The tracheary elements (TEs) of the xylem serve as the water‐conducting vessels of the plant vascular system. To achieve this, TEs undergo secondary cell wall thickening and cell death, during which the cell contents are completely removed. Cell death of TEs is a typical example of developmental programmed cell death that has been suggested to be autophagic. However, little evidence of autophagy in TE differentiation has been provided. The present study demonstrates that the small GTP binding protein RabG3b plays a role in TE differentiation through its function in autophagy. Differentiating wild type TE cells were found to undergo autophagy in an Arabidopsis culture system. Both autophagy and TE formation were significantly stimulated by overexpression of a constitutively active mutant (RabG3bCA), and were inhibited in transgenic plants overexpressing a dominant negative mutant (RabG3bDN) or RabG3b RNAi (RabG3bRNAi), a brassinosteroid insensitive mutant bri1‐301, and an autophagy mutant atg5‐1. Taken together, our results suggest that autophagy occurs during TE differentiation, and that RabG3b, as a component of autophagy, regulates TE differentiation.