低温胁迫下草菇甘油‐3‐磷酸酰基转移酶基因表达变化的研究

以草菇低温敏感型V23菌株与耐低温型VH3菌株为试验材料,将二者菌丝体置于冰浴中进行不同时间的低温胁迫处理。首先提取RNA,反转录为cDNA,然后构建含有微管蛋白(tubulin,TUB)基因片段和甘油‐3‐磷酸酰基转移酶(glycerol‐3‐phosphate acyltransferase,GPAT)基因片段的质粒,最终对GPAT基因在低温胁迫不同处理时间下的表达进行定量。结果表明,耐低温型的VH3菌株,在低温处理2h时,GPAT基因相对表达量上升,4h,表达量下降,6h上升,之后逐渐下降。低温敏感型V23菌株,在低温处理2h时,表达量下降,4h,表达量上升,此后逐渐下降;除低温处理4h外,V23菌株的GPAT基因表达量始终低于VH3菌株,初步推测GPAT基因的高表达与草菇的耐低温能力相关。     

白鹅催乳素基因的克隆及诱导表达条件的优化

摘要: 运用RT-PCR方法, 从白鹅脑垂体总RNA中扩增得到了催乳素(Prolactin, PRL)基因编码区序列cDNA, 并将其克隆到pMD18-T载体上。DNA序列分析表明, PRL cDNA包括终止密码子在内的长度为690 bp,编码230个氨基酸残基的蛋白质, 与皖西白鹅的有所差异, 二者碱基同源性在99.57%, 氨基酸同源性达99.56%。将PRL基因编码区序列cDNA定向克隆到表达载体pET-32a (+)中, 构建表达质粒pET-32a(+)-PRL。该质粒的BL21 (DE3)转化菌在IPTG的诱导下可表达PRL基因融合蛋白, IPTG终浓度1 mmol/L, 37℃, 诱导4 h表达量最高, 表达量约占菌体总蛋白的28.96%。     

瓜实蝇热激蛋白Hsp90基因克隆及表达分析

【目的】热激蛋白基因Hsps在生物体抗逆性过程中具有重要的作用,本文旨在通过阐释瓜实蝇Bactrocera cucurbitae (Coquillett)热激蛋白基因Hsp90在其对环境温度适应性及抗药性过程中的功能作用,为瓜实蝇综合治理提供理论基础。【方法】采用RACE-PCR技术克隆瓜实蝇的Hsp90基因cDNA序列,利用生物信息学工具分析序列特征,并通过实时定量PCR技术分析高温胁迫、阿维菌素诱导和抗性及敏感品系中该基因的表达情况。【结果】克隆获得的瓜实蝇Hsp90基因cDNA全长2 654 bp,命名为Bc-Hsp90,GenBank登录号为KP864677,含2 145 bp的开放阅读框,编码715个氨基酸蛋白,具有HSP90蛋白家族共有模式和1个基序标签,C-末端具有MEEVD基序,属于胞质型热激蛋白。系统发育分析显示,Bc-Hsp90基因高度保守。不同高温(32-40℃)胁迫处理1 h和2 h后,瓜实蝇成虫体内Bc-Hsp90的相对表达量均显著高于对照组(26℃),并在38℃和40℃表达量最高;阿维菌素长期筛选的RS品系,Bc-Hsp90表达量是对照SS品系的2.13倍,但RS品系以LC90剂量诱导后,Bc-Hsp_(90)表达量显著下调,随着恢复时间延长其表达量逐渐升高,96 h后恢复到对照水平。【结论】推测Bc-Hsp90在瓜实蝇耐热性和抗阿维菌素过程中可能起着重要作用。     

湖北海棠MhPR1a基因的克隆与表达特性分析

以水杨酸诱导的湖北海棠[ Malus hupehensis (Pamp.) Rehd.]全长cDNA文库和基因组DNA为模板,克隆其PR1a基因(MhPR1a)的全编码区序列,并对该序列进行生物信息学分析;在此基础上利用荧光定量RT-PCR技术对湖北海棠根、茎和叶中该基因的表达特性及经过10μmol·L-1ABA、4℃低温处理及苹果蚜虫(Aphis citricola van der Goot)侵染后叶中该基因的表达特性进行了测定.结果表明:克隆获得的MhPR1a基因全长518 bp,最大开放阅读框为492 bp,编码162个氨基酸残基;编码的蛋白质为酸性蛋白,其相对分子质量为16 960,等电点pI 5.46;其基因组DNA序列与cDNA序列完全一致,说明MhPR1a基因内部没有内含子.湖北海棠MhPR1a基因与苹果(M.domestic Borkh.)和沙梨[Pyrus pyrifolia( Burm.f.)Nakai] PR1基因的cDNA序列及其编码的氨基酸序列同源性均较高,其中cDNA序列的同源性均为97％,氨基酸序列的同源性分别为95％和97％;系统树也显示MhPR1a基因编码的氨基酸序列与苹果和沙梨的亲缘关系最近,聚为一类.MhPR1a基因编码的氨基酸序列具有SCP保守结构域,含有1个信号肽和6个保守的半胱氨酸残基.在湖北海棠的叶、茎和根中MhPR1a基因均能表达,在根中的表达量最高.10 μmol·L-1ABA和4℃低温处理48 h后均可诱导MhPR1a基因的表达,且相对表达量明显高于对照(处理0h);苹果蚜虫也可诱导MhPR1a基因的表达,说明MhPR1a基因在湖北海棠抵抗植食昆虫和低温胁迫的过程中可能发挥着重要作用.     

东方百合LoABI1基因的克隆及表达分析

该研究以东方百合‘索邦’为材料,采用RT-PCR扩增方法克隆ABI1基因,对其进行了生物信息学分析,并采用实时荧光定量PCR检测其组织表达特性和低温处理过程及定植后的表达特征,以明确ABI1基因的功能特性,为解析百合ABA信号转导途径及其调控低温解除休眠过程的机理奠定基础。结果表明：(1)成功克隆得到东方百合LoABI1基因,其编码序列长度为1 341 bp,共编码446个氨基酸;LoABI1氨基酸序列中含有1个蛋白磷酸酶2C(PP2C)保守结构域。(2)系统进化分析显示,LoABI1蛋白与水稻PP2C家族成员OsPP2C06的同源进化关系最近,且与拟南芥AtABI1聚为一支,同属于PP2C基因家族中的A亚群。(3)亚细胞定位发现,LoABI1蛋白定位于烟草表皮细胞的细胞核和细胞质。(4)qRT-PCR荧光定量分析显示,LoABI1基因在百合茎生根、嫩茎、叶片及各花部组织中均有表达,且在幼嫩组织中表达量较高;LoAB1I基因在冷藏期间的表达量呈先升高后降低的趋势,并于冷藏期第5周达到峰值,但在定植期间持续下降且保持较低水平。(5)经4℃低温处理60 d的百合鳞茎在定植后14～28 d能...     

黄瓜(Cucumis stavius L.)CSEBF1基因的克隆及其表达特性分析

通过对乙烯利诱导黄瓜茎尖SSH文库的筛选,采用RT-PCR和电子克隆技术,从黄瓜中克隆到一个EIN3-Binding F box protein 1基因,命名为CSEBF1基因(GenBank登录号为KF366911)。结果表明:CSEBF1基因的cDNA全长1 964bp,编码640个氨基酸,含有F-box蛋白保守区域和蛋白质泛素化作用底物识别必需结构—LRRs(leucine-rich repeats),氨基酸序列与拟南芥的同源性为60.47%。实时荧光定量RT-PCR法分析了CSEBF1基因在乙烯利诱导后植株不同部位的表达情况,表明该基因在叶片和根部处理8 h达到最高值,而在茎部16 h达到最高值。同时通过RTPCR方法检测茎、叶部乙烯信号转导相关基因的表达情况发现,CSEIN3基因于处理4 h在茎、叶诱导表达;CSCTR1基因在处理后8 h的茎和16 h的叶片有表达量;CSACS2基因在处理后2 h的叶片被诱导表达,并且表达量随后增加。CSACS1G基因(即F基因)于处理后4 h的叶片增强表达,随后持续较微弱的水平。     

西伯利亚蓼非特异性脂质转移蛋白编码序列的克隆及其盐胁迫下的表达

根据西伯利亚蓼地下茎抑制消减文库(SSH)中获得的非特异性脂质转移蛋白(non-specific lipid transfer protein, nsLTP)EST序列,应用RACE技术克隆了具有Poly A的全长cDNA序列.该序列全长604 bp,其5′非翻译区65 bp,3′非翻译区227 bp,开放阅读框编码103个氨基酸残基;序列分析表明,该基因具有N端信号肽,具有nsLTP家族共有的典型保守区域,属nsLTP家族基因,命名为PsnsLTPs;荧光定量PCR分析表明,PsnsLTPs在西伯利亚蓼叶、茎、地下茎中均有表达.在3%NaHCO3诱导表达下,该基因在地下茎中表达明显受盐胁迫的诱导,推测该基因在抵御盐胁迫时具有重要作用.     

玉米生物钟基因ZmPRR1-2的克隆及表达分析

为探究玉米生物钟基因ZmPRR1-2的功能及表达特性，解析玉米光周期途径调控开花的机理，该研究以玉米骨干自交系‘黄早4’为材料，克隆ZmPRR1-2基因的cDNA序列并进行生物信息学分析；利用qRT-PCR技术对该基因进行组织特异性表达分析和48 h的昼夜节律表达分析。结果表明：(1)成功克隆获得ZmPRR1-2基因的编码区全长1 554 bp,编码517个氨基酸，编码的蛋白属于PRR基因家族，含有1个REC结构域和1个CCT结构域，多序列比对和系统进化分析显示ZmPRR1-2基因在禾本科植物中高度保守；ZmPRR1-2蛋白属亲水性蛋白，不包含跨膜结构域和信号肽。(2)ZmPRR1-2基因在玉米叶片中的表达量最高，显著高于其他7个组织，表明该基因主要在叶片中发挥功能，而在果穗和花丝中表达量相对较低，且显著低于雄穗中的表达量。(3)昼夜节律表达分析显示，在短日照条件下，ZmPRR1-2基因的表达量于光照3 h后开始逐渐上升，在光照结束后3 h时达到表达高峰；在长日照条件下，于光照6 h后ZmPRR1-2基因的表达量才开始逐渐上升，且在光照结束时达到表达高峰。研究认为，ZmPRR1-2基因...     

蝴蝶兰PhTSJT1基因的克隆及在低温胁迫下的表达分析

本研究从蝴蝶兰叶片中克隆了茎部特异基因Ph TSJT1的全长序列(GenBank登录号为MF797883),并分析了其在不同组织及低温胁迫下的表达特性。结果表明,Ph TSJT1基因全长994 bp,编码239个氨基酸,属于Class-Ⅱ谷氨酰胺酰胺基转移酶超家族成员;同源性分析表明,该蛋白与多种植物的茎部特异蛋白和铝诱导蛋白有较高的同源性,进化上与小兰屿蝴蝶兰的茎部特异蛋白亲缘关系最近;该基因在营养器官中表达水平较高,在花器官中表达水平较低;13℃/8℃(昼/夜)的低温胁迫抑制PhTSJT1基因的转录表达,并随着低温胁迫时间的延长,Ph TSJT1基因的表达水平逐渐降低,在温度恢复正常时其表达水平升高;4℃冷胁迫低温条件下,PhTSJT1基因在处理1 h时,表达水平升高,处理8 h时表达水平最高,16 h后表达水平逐渐降低。由此推测,PhTSJT1参与4℃冷胁迫的分子调控。本研究不但有助于理解热带亚热带植物的耐冷机制,也为蝴蝶兰新品种的遗传改良提供帮助。     

芜菁BrrHMA2.1和BrrHMA2.2基因的克隆与表达

该研究以芜菁(Brassica rapa var.rapa)为材料,克隆得到重金属ATP酶(HMA)家族1对同源基因BrrHMA2.1(GenBank登录号:MG_283237)和BrrHMA2.2(GenBank登录号:MG_283238),并对其蛋白质序列特征和基因表达模式进行分析。结果表明:(1)BrrHMA2.1和BrrHMA2.2基因的全长开放阅读框分别为2 619和2 724bp,分别编码872和907个氨基酸;序列结构分析显示,BrrHMA2.1和BrrHMA2.2蛋白含有6个跨膜区和HMA蛋白家族保守结构域;系统进化树结果显示,BrrHMA2.1和BrrHMA2.2蛋白与拟南芥HMA家族成员AtHMA2进化关系最近。(2)亚细胞定位结果表明,BrrHMA2.1和BrrHMA2.2蛋白都定位于细胞膜上。(3)qRT-PCR分析表明,芜菁生长初期BrrHMA2.1和BrrHMA2.2基因在叶中的表达量最高;随着生长时间的延长,叶中的表达量逐渐降低,而根中的表达量逐渐增加。(4)研究发现,BrrHMA2.1受Cd~(2+)、Zn~(2+)、Na~+、Mg~(2+)胁迫诱导表达,BrrHMA2.2受Cd~(2+)、Na~+、Cu~(2+)胁迫诱导表达,表明2个基因可能参与这些金属离子的转运过程。该研究结果为进一步研究植物HMA基因在重金属吸收和转运过程中的功能奠定了基础。     

Rat sn-glycerol-3-phosphate acyltransferase: molecular cloning and characterization of the cDNA and expressed protein.

Rat mitochondrial glycerol-3-phosphate acyltransferase (GPAT) cDNA was cloned and characterized. We identified a cDNA containing an open reading frame of 828 amino acids that had an 89% homology with the coding region of the previously characterized mouse mitochondrial GPAT and a predicted amino acid sequence that was 96% identical. The rat 5' UTR was only 159 nucleotides, in contrast to the 926 nucleotide 5' UTR of the mouse cDNA and had an internal deletion of 167 nucleotides. GPAT was expressed in Sf21 insect cells, and specific inhibitors strongly suggest that, like the Escherichia coli GPAT, the recombinant mitochondrial GPAT and the mitochondrial GPAT isoform in rat liver contain critical serine, histidine, and arginine residues.     

The C-terminal region of mitochondrial glycerol-3-phosphate acyltransferase-1 interacts with the active site region and is required for activity

Glycerol phosphate acyltransferase (GPAT) catalyzes the formation of 1-acyl-sn-glycerol-3-phosphate from glycerol-3-phosphate and long chain fatty acyl-CoA substrates. We previously determined the topography of the mitochondrial GPAT1 isoform (mtGPAT1, 828 amino acids). mtGPAT1 has two transmembrane domains (TMDs) (aa 472-493 and aa 576-592) with both the N- and C-termini facing the cytosol and a loop (aa 494-575) facing the intermembrane space. Alignment of amino acid sequences from mtGPAT1 and other acyltransferases and site directed mutagenesis studies have demonstrated that the active site of the enzyme resides in the N-terminal domain of the protein. In this study, we sequentially truncated the C-terminal domain and characterized the properties of the resulting mutants expressed in CHO cells. Although the mutants were overexpressed, none of them conferred GPAT activity. The loss of activity was not due to the miss-targeting of the proteins since immunofluorescence experiments demonstrated their mitochondrial localization. Instead, chemical crosslinking and protein cleavage studies demonstrated that the N- and C-termini of the protein interact. These results suggest that the C-terminal domain is necessary for mtGPAT1 activity, and probably contributes to catalysis or substrate binding.     

Cloning and characterization of GPAT gene from Lepidium latifolium L.: a step towards translational research in agri-genomics for food and fuel

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes first and the rate limiting step in glycerolipid synthesis pathway, which in turn contribute to stabilization of plasma membrane structure and oil lipid synthesis in plant cells. Here, we report cloning and characterization of GPAT gene from Lepidium latifolium (LlaGPAT). The cDNA sequence (1,615 bp) of LlaGPAT gene consisted of 1,113 bp ORF encoding a protein of 370 aa residues, with deduced mass of 41.2 kDa and four acyltransferase (AT) motifs having role in catalysis and in glycerol-3-phosphate binding. Southern blot analysis suggested presence of a single copy of the gene in the genome. Tissue specific expression of the gene was seen more abundantly in aerial parts, compared to the roots. Quantitative real-time PCR indicated down-regulation of the gene by cold (4 °C), drought (PEG6000), salt (300 mM NaCl) and ABA (100 μM) treatments. Considering the vitality of the function of encoded enzyme, LlaGPAT can be considered a potential candidate gene for genetic engineering of oil yields and abiotic stress management in food as well as fuel crops.     

Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction

Alignment of amino acid sequences from various acyltransferases [sn-glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), acyl-CoA:dihydroxyacetone-phosphate acyltransferase (DHAPAT), 2-acylglycerophosphatidylethanolamine acyltransferase (LPEAT)] reveals four regions of strong homology, which we have labeled blocks I-IV. The consensus sequence for each conserved region is as follows: block I, [NX]-H-[RQ]-S-X-[LYIM]-D; block II, G-X-[IF]-F-I-[RD]-R; block III, F-[PLI]-E-G-[TG]-R-[SX]-[RX]; and block IV, [VI]-[PX]-[IVL]-[IV]-P-[VI]. We hypothesize that blocks I-IV and, in particular, the invariant amino acids contained within these regions form a catalytically important site in this family of acyltransferases. Using Escherichia coli GPAT (PlsB) as a model acyltransferase, we examined the role of the highly conserved amino acid residues in blocks I-IV in GPAT activity through chemical modification and site-directed mutagenesis experiments. We found that the histidine and aspartate in block I, the glycine in block III, and the proline in block IV all play a role in E. coli GPAT catalysis. The phenylalanine and arginine in block II and the glutamate and serine in block III appear to be important in binding the glycerol 3-phosphate substrate. Since blocks I-IV are also found in LPAAT, DHAPAT, and LPEAT, we believe that these conserved amino acid motifs are diagnostic for the acyltransferase reaction involving glycerol 3-phosphate, 1-acylglycerol 3-phosphate, and dihydroxyacetone phosphate substrates.     

Glycerol-3-phosphate acyltransferase-2 is expressed in spermatic germ cells and incorporates arachidonic Acid into triacylglycerols

Background

De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. In this work we show that mitochondrial GPAT2 overexpression in CHO-K1 cells increased TAG content and both GPAT and AGPAT activities 2-fold with arachidonoyl-CoA as a substrate, indicating specificity for this fatty acid.

Methods and Results

Incubation of GPAT2-transfected CHO-K1 cells with [1-¹⁴C]arachidonate for 3 h increased incorporation of [¹⁴C]arachidonate into TAG by 40%. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2''s role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein.

Conclusions

These results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.     

Lysophosphatidic acid activates peroxisome proliferator activated receptor-γ in CHO cells that over-express glycerol 3-phosphate acyltransferase-1

Lysophosphatidic acid (LPA) is an agonist for peroxisome proliferator activated receptor-γ (PPARγ). Although glycerol-3-phosphate acyltransferase-1 (GPAT1) esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO) cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA) or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression.     

Enhancement of seed oil content by expression of glycerol-3-phosphate acyltransferase genes

Arabidopsis thaliana was transformed with a plastidial safflower glycerol-3-phosphate acyltransferase (GPAT) and an Escherichia coli GPAT. The genes were used directly and in modified forms with, as applicable, the plastidial targeting sequence removed, and with an endoplasmic reticulum targeting sequence added. Seeds of plants transformed using only the vector were indistinguishable in oil content from wild-type control plants. All other gene constructs increased seed oil content. The unmodified safflower gene (spgpat) produced oil increases ranging from 10 to 21%. On average, the greatest increase (+22%) was observed in seeds of transformants carrying the spgpat with the targeting peptide removed. The E. coli plsB gene increased seed oil content by an average of 15%.     

Evidence for interactions of acyl carrier protein with glycerol-3-phosphate acyltransferase, an inner membrane protein of Escherichia coli

We [(1989) FEBS Lett., in press] have previously shown that membrane vesicles from Escherichia coli contain protein-binding sites for the acyl carrier protein (ACP). We report now that membrane vesicles prepared from a strain amplified for glycerol-3-phosphate acyltransferase (GPAT) contain a higher number of ACP-binding sites than the membrane vesicles prepared from a wild type strain. In addition, we show that GPAT is retained specifically on an ACP-Sepharose affinity column and that [3H]ACP binds to the enzyme solubilized by detergent. We conclude that GPAT, an inner membrane protein which catalyses the transesterification of a fatty acyl group from acyl coenzyme A or acyl ACP to glycerol-3-phosphate, possesses a binding site for ACP.