一类新的编码PRPs基因的分离及其在棉花纤维等组织细胞中的表达

富含脯氨酸的细胞壁蛋白(proline-rich proteins,PRPs)在植物中广泛分布,它们在建造围绕特定细胞类型的细胞壁结构上起着很重要的作用.从棉花的cDNA文库中分离了5个编码富含脯氨酸的细胞壁蛋白质基因,这5个基因推断的氨基酸序列最普遍的特点就是脯氨酸含量非常高.根据氨基酸组成、富含脯氨酸的重复单元和结构域组织的特点,将这5个蛋白质分成2个亚类:一类(包括GhPRP3-6)与典型的PRPs结构相似,由N端疏水区(或信号肽)与不同富含脯氨酸的重复序列组成;另一类(GhPRPL)与典型的PRPs结构不同,这个蛋白质的N端为亲水序列,GhPRPL在靠近C端有8个5肽(类似PPKKE)的重复基序,与典型PRPs所含有的重复序列PPVYK非常相似.实时RT-PCR(Real-time RT-PCR)分析表明,GhPRP3和GhPRP5在10dpa纤维中特异表达,而GhPRPL在子叶中优势表达.GhPRP4和GhPRP6在所分析的组织中都有表达,GhPRP4mRNA在下胚轴中最丰富,在花药中次之,而GhPRP6在10dpa纤维中表达最强,在10dpa胚珠中次之.此外,GhPRP3,GhPRP5基因表达受纤维发育调节,表明它们可能在棉纤维发育中起重要作用.     

草原龙胆MADS-box基因的克隆及表达分析

植物MADS-box基因家族编码高度保守的转录因子,参与了包括花发育在内的多种发育进程。为阐释双子叶植物草原龙胆（Eustoma grandiflorum）花器官发育的分子调控机制,根据MADS-box基因保守序列设计简并引物,用3＇-RACE方法从草原龙胆中克隆了4个花器官特异表达的MADS-box家族基因。序列和系统进化树分析表明,这4个基因分别与金鱼草DEF基因、矮牵牛FBP3基因和FBP6基因以及拟南芥SEP3基因具有很高的同源性,分别属DEF/GLO、AG-like和SEP-like亚家族。从而将这4个基因分别命名为EgDEF1、EgGLO1、EgPLE1和EgSEP3-1。推导的氨基酸序列显示,这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域,每个基因均有其亚家族特异的C-末端功能域。基因特异性RT-PCR检测结果显示：EgDEF1在萼片、花瓣、雄蕊及胚珠中高丰度表达,在心皮中微量表达;而EgGLO1在花瓣和雄蕊中高丰度表达,在萼片中微量表达;在根、茎、叶等营养器官中均未检测到上述2个基因的表达。EgPLE1在雌蕊、心皮和胚珠中特异表达,但表达的丰度存在差异,在雄蕊中的表达有所减弱。SEP-like亚家族基因EgSEP3-1在四轮花器官和胚珠中均特异表达,且表达丰度相对一致。     

草原龙胆MADS-box基因的克隆及表达分析

植物MADS-box 基因家族编码高度保守的转录因子, 参与了包括花发育在内的多种发育进程。为阐释双子叶植物草原龙胆(Eustoma grandiflorum)花器官发育的分子调控机制, 根据MADS-box基因保守序列设计简并引物, 用3'-RACE方法从 草原龙胆中克隆了4个花器官特异表达的MADS-box家族基因。序列和系统进化树分析表明, 这4个基因分别与金鱼草DEF基因、矮牵牛FBP3基因和FBP6基因以及拟南芥SEP3基因具有很高的同源性, 分别属DEF/GLO、AG-like和SEP-l ike亚家族。从而将这4个基因分别命名为EgDEF1、EgGLO1、EgPLE1和EgSEP3-1。推导的氨基酸序列显示, 这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域, 每个基因均有其亚家族特异的C-末端功能域。基因特异性RT-PCR检测结果显示: EgDEF1 在萼片、花瓣、雄蕊及胚珠中高丰度表达, 在心皮中微量表达; 而EgGLO1在花瓣和雄蕊中高丰度表达, 在萼片中微量表达; 在根、茎、叶等营养器官中均未检测到上述2个基因的表达。EgPLE1在雌蕊、心皮和胚珠中特异表达, 但表达的丰度存在差异, 在雄蕊中的表达有所减弱。SEP-like亚家族基因EgSEP3-1在四轮花器官和胚珠中均特异表达,且表达丰度相对一致。     

大豆斑疹病菌harpin编码基因的克隆与特性研究

根据黄单胞菌harpin编码基因的同源性,设计简并引物,采用PCR方法从大豆斑疹病菌（Xanthomonas axonopodis pv.glycines, Xag）中克隆了402 bp的[STBX]hpa1[STBZ]同源基因,构建于表达载体pET30(a)上经转化大肠杆菌BL21菌株,获得基因工程菌BHR_3。基因工程菌诱导表达后经收集菌体和破碎细胞,得到表达产物为151kD的蛋白质。该蛋白质富含甘氨酸,不含半胱氨酸,对热稳定,对蛋白酶K敏感,可在非寄主烟草上激发过敏反应。激发的过敏反应需要植物体内水杨酸的积累,还可被真核生物代谢抑制剂抑制。序列比较显示,该基因与Xag中hpaG基因相同,与其它黄单胞菌中的hpa1基因有51.4%～93.8%的同源性,与其它革兰氏阴性植物病原细菌的harpin编码基因无同源性。据此把该基因产物鉴定为harpinXag。黄单胞菌harpin蛋白质序列比较发现,GG_GGG基序的多少并不是harpin蛋白的唯一特性。这为利用harpin蛋白开展植物病害控制的基因药物学设计提供了科学线索。     

棉花LIM结构域基因(GhLIM1)的克隆和表达分析

LIM结构域蛋白是一个重要的发育调控因子,参与基因转录,细胞骨架建成和信号传导等许多发育调控过程,胞质骨架是形成和稳定细胞形态以及传递物质,能量和信息的重要成分。为研究棉花纤维细胞发育过程中胞质骨架的形成和作用机理,通过棉花纤维EST序列整合,从陆地棉徐州142胚珠（含纤维）中扩增并克隆出棉花LIM结构域基因的编码区段。该棉花LIM结构域基因（GhL1M1)长848bp,包含一个570bp的开放阅读框,推导的氨基酸序列（189个氨基酸）与拟南芥,烟草和向日葵的LIM结构域蛋白有极高的同源性,而且两个LIM结构域完整,RT-PCR和Northerm杂交分析表明,该基因（GhL1M1）在陆地棉的根,茎尖,上胚轴,叶片,花蕾,花药,胚珠和不同发育时期的陆地棉纤维（4DPA、12DPA、18DPA）以及海岛棉纤维（18DPA）和中棉纤维（12DPA）中均有表达,但GhL1M1基因在茎尖,纤维和有纤维的胚珠中表达量更高,因此GhL1M1基因应与棉花纤维发育有密切关系。     

水稻Osgrp-2基因的结构、表达特性和染色体定位

富含甘氨酸的蛋白质(GRP)是植物细胞壁的一种重要结构蛋白, GRP基因的表达具有组织特异性, 受发育阶段和多种环境因素的调控, 因此GRP基因为植物基因的表达调控研究提供了一个良好的模式. 从水稻基因组文库中克隆了一个新的GRP基因Osgrp-2, 测定了其序列, 并通过5′RACE实验确定了该基因的转录起始位点, 从而界定了约2.4 kb的启动子序列. 还详细分析了Osgrp-2在水稻中的时空表达特性和损伤诱导表达特性. 最后, 将Osgrp-2定位于水稻的第10号染色体上.     

三叶因子家族

三叶因子家族是一类具有特殊结构——P结构域的蛋白质家族, P结构域包含6个高度保守的半胱氨酸残基及精氨酸、甘氨酸、色氨酸和苯丙氨酸残基.半胱氨酸残基以Cys1-Cys5, Cys2-Cys4, Cys3-Cys6连接形成三个链内二硫桥.已发现多种含有P结构域的多肽,其中最引人注目的是TFF1/ pS2、TFF2/SP及TFF3/ITF,在正常组织中其主要表达位点分别为胃基底和胃体(TFF1/pS2)、胃窦深部的小凹（TFF2/SP）及小肠和大肠杯状细胞（TFF3/ITF）.TFF可能具有维持粘膜屏障和促进溃疡治愈的功能.TFF含有α折叠(α-helix),β片层(β-sheet).三叶因子家族多肽的作用机理仍处于猜测阶段,现有与粘蛋白共同作用和与受体作用两种假说.     

转GFP::cDNA基因拟南芥筛选及相关基因的功能分析

本研究以随机GFP::cDNA融合基因转基因拟南芥为材料,筛选到在细胞核或在细胞核和细胞质中均表达GFP信号的转基因株系58个。对这些转基因株系中的cDNA插入片段进行克隆,获得4株插入片段能按原初编码框进行编码的转基因株系。对插入片段为编码富含甘氨酸蛋白AtGRP8 C-末端(富含甘氨酸的结构域)的转基因株系R2的表型分析发现,连续白光、红光或蓝光下其幼苗的下胚轴比野生型的要短,且较低光照强度白光(低于100μmol m-2s-1)、蓝光(低于75μmol m-2s-1)下的差异更加明显,但是黑暗中其幼苗的下胚轴与野生型相比无明显差异,表明AtGRP8蛋白可能通过其C-末端功能域参与调控拟南芥的光形态建成反应。     

真核蛋白质中G-patch结构域的结构和功能研究

G-patch是广泛存在真核生物蛋白质中的保守结构域,含有六个高度保守的甘氨酸残基,推测它是一种RNA结合结构域,其功能与RNA代谢有关.     

陆地棉SUPERMAN类似锌指蛋白基因的克隆与表达分析

锌指蛋白是生物体内数量最多的转录调控因子,它在动植物的生长发育中都起到十分重要的作用。SUPERMAN类锌指蛋白只含有1个锌指结构。我们根据这类蛋白的保守结构域设计简并引物,通过RT-PCR从棉花中获得了3个这个家族成员的EST,得到1个锌指蛋白基因的全长序列,该基因的编码区长744 bp,编码长248个氨基酸的多肽,其氨基酸序列与GenBank中登录的一个拟南芥RBE蛋白有40%的同源性。此基因被命名为GZFP。它含有保守的锌指结构并在多肽链的C-端具有富含亮氨酸的保守结构域,GZFP含有核定位信号并且没有内含子。GZFP基因在棉花花蕾、子房、花瓣和根中的表达量要高于木质部、韧皮部、叶片、纤维和种子。GZFP基因的表达量很低,在GenBank中没有任何和它同源的EST序列存在。对GZFP 5′侧翼区进行分析发现有数个花粉和根特异表达相关元件,4个与Dof蛋白作用的核心序列,4个与光诱导相关的元件。      

棉花143-3L基因的分子鉴定及其在纤维发育中优势表达分析

14-3-3蛋白以二聚体形式存在于所有真核生物中,是一种高度保守的调节蛋白,在细胞生长、增殖、凋亡、信号转导等生命活动中发挥着重要调控作用。我们在棉纤维cDNA文库中分离克隆到1个基因(cDNA),编码14-3-3蛋白类似物,命名为Gh14-3-3L(Gossypiumhirsutum14-3-3-like)。该cDNA长度为1,029bp,包含762bp开放阅读框,其编码蛋白由253个氨基酸组成。Gh14-3-3L与其他真核生物的14-3-3蛋白具有较高的同源性,并具有14-3-3蛋白的基本结构:二聚体结构域、磷酸化丝氨酸富集识别序列、4个CC结构和1个EFHand结构。Northern杂交分析显示Gh14-3-3L在棉纤维发育早期优势表达,且在10DPA棉纤维细胞中表达量最高,这表明Gh14-3-3L基因可能涉及棉纤维细胞伸长过程的调节。研究还表明,该基因在胚珠和花瓣组织中也有较强的表达,但在其他组织中表达较弱或不表达。     

一个陆地棉bZIP蛋白cDNA的克隆及表达分析

利用PCR筛选方法从陆地棉纤维cDNA文库中筛选到一个全长cDNA序列,命名为GhbZIP。其编码产物长度为645个氨基酸残基,序列中含有两个未知功能的保守区域DUF630和DUF632,而DUF632区中有一个类似碱性亮氨酸拉链基元;此外氨基酸序列中还存在一个富脯氨酸区和一个富苯丙氨酸区,因此该蛋白具有植物碱性亮氨酸拉链蛋白的结构特征。亲水性分析表明,GhbZIP为一个典型的膜蛋白。GhbZIP基因主要是在开花3d之后在胚珠和纤维细胞中表达,这表明该基因可能与棉纤维伸长过程中的基因表达调控有关。     

The peroxisomal membrane targeting elements of human peroxin 2 (PEX2)

Peroxin 2 (PEX2) is a 35-kDa integral peroxisomal membrane protein with two transmembrane regions and a zinc RING domain within its cytoplasmically exposed C-terminus. Although its role in peroxisome biogenesis and function is poorly understood, it seems to be involved in peroxisomal matrix protein import. PEX2 is synthesized on free cytosolic ribosomes and is posttranslationally imported into the peroxisome membrane by specific targeting information. While a clear picture of the basic targeting mechanisms for peroxisomal matrix proteins has emerged over the past years, the targeting processes for peroxisomal membrane proteins are less well understood. We expressed various deletion constructs of PEX2 in fusion with the green fluorescent protein in COS-7 cells and determined their intracellular localization. We found that the minimum peroxisomal targeting signal of human PEX2 consists of an internal protein region of 30 amino acids (AA130 to AA159) and the first transmembrane domain, and that adding the second transmembrane domain increases targeting efficiency. Within the minimum targeting region we identified the motif "KX6(I/L)X(L/F/I)LK(L/F/I)" that includes important targeting information and is also present in the targeting regions of the 22-kDa peroxisomal membrane protein (PMP22) and the 70-kDa peroxisomal membrane protein (PMP70). Mutations in this targeting motif mislocalize PEX2 to the cytosol. In contrast, the second transmembrane domain does not seem to have specific peroxisomal membrane targeting information. Replacing the second transmembrane domain of human PEX2 with the transmembrane domain of human cytochrome c oxidase subunit IV does not alter PEX2 peroxisome targeting function and efficiency.     

Molecular cloning and expression of the C-terminus of spider flagelliform silk protein from <Emphasis Type="Italic">Araneus ventricosus</Emphasis>

A cDNA coding for the C-terminus of spider flagelliform silk protein (AvFlag) was cloned from Araneus ventricosus. Analysis of the cDNA sequence shows that the C-terminus of AvFlag consists of 167 amino acids of a repetitive region and 87 amino acids of a C-terminal non-repetitive region. The peptide motifs found in spider flagelliform silk proteins, GPGGX and GGX, were conserved in the repetitive region of AvFlag. Phylogenetic analysis further confirmed that AvFlag belongs to the spider flagelliform silk proteins. The AvFlag cDNA was expressed as a 28 kDa polypeptide in baculovirus-infected insect cells. As a new expression approach for spider silk protein, the combination of polyhedrin and AvFlag creates a polyhedrin AvFlag fusion protein (61 kDa) that is produced as recombinant polyhedra; this provides a basis for the source of spider silk proteins for various applications.     

Three new members of the RNP protein family in Xenopus.

Many RNP proteins contain one or more copies of the RNA recognition motif (RRM) and are thought to be involved in cellular RNA metabolism. We have previously characterized in Xenopus a nervous system specific gene, nrp1, that is more similar to the hnRNP A/B proteins than to other known proteins (K. Richter, P. J. Good, and I. B. Dawid (1990), New Biol. 2, 556-565). PCR amplification with degenerate primers was used to identify additional cDNAs encoding two RRMs in Xenopus. Three previously uncharacterized genes were identified. Two genes encode hnRNP A/B proteins with two RRMs and a glycine-rich domain. One of these is the Xenopus homolog of the human A2/B1 gene; the other, named hnRNP A3, is similar to both the A1 and A2 hnRNP genes. The Xenopus hnRNP A1, A2 and A3 genes are expressed throughout development and in all adult tissues. Multiple protein isoforms for the hnRNP A2 gene are predicted that differ by the insertion of short peptide sequences in the glycine-rich domain. The third newly isolated gene, named xrp1, encodes a protein that is related by sequence to the nrp1 protein but is expressed ubiquitously. Despite the similarity to nuclear RNP proteins, both the nrp1 and xrp1 proteins are localized to the cytoplasm in the Xenopus oocyte. The xrp1 gene may have a function in all cells that is similar to that executed by nrp1 specifically within the nervous system.     

Cytochrome b561 protein family: expanding roles and versatile transmembrane electron transfer abilities as predicted by a new classification system and protein sequence motif analyses

Cytochrome b561 family was characterized by the presence of "b561 core domain" that forms a transmembrane four helix bundle containing four totally conserved His residues, which might coordinate two heme b groups. We conducted BLAST and PSI-BLAST searches to obtain insights on structure and functions of this protein family. Analyses with CLUSTAL W on b561 sequences from various organisms showed that the members could be classified into 7 subfamilies based on characteristic motifs; groups A (animals/neuroendocrine), B (plants), C (insects), D (fungi), E (animals/TSF), F (plants+DoH), and G (SDR2). In group A, both motif 1, {FN(X)HP(X)2M(X)2G(X)5G(X)ALLVYR}, and motif 2, {YSLHSW(X)G}, were identified. These two motifs were also conserved in group B. There was no significant features characteristic to groups C and D. A modified version of motif 1, {LFSWHP(X)2M(X)3F(X)3M(X)EAIL(X)SP(X)2SS}, was found in group E with a high degree of conservation. Both motif 3, {DP(X)WFY(L)H(X)3Q}, and motif 4, {K(X)R(X)YWN(X)YHH(X)2G(R/Y)} ,were found in group F at different regions from those of motifs 1 and 2. The "DoH" domain common to the NH2-terminal region of dopamine beta-hydroxylase was found to form fusion proteins with the b561 core domains in groups F and G. Based on these results, we proposed a hypothesis regarding structures and functions of the 7 subfamilies of cytochrome b561.