小麦泛素融合降解蛋白基因的克隆及特征分析

酵母UFD1基因编码的泛素融合降解蛋白是泛素依赖性降解系统或泛素融合降解途径中的一个关键因子。利用RT-PCR技术在小麦(Triticum aestivum L.)中分离到一个UFD1类似基因。该基因的编码区长948 bp,编码长315个氨基酸的多肽,其氨基酸序列与GenBank中登录的一个拟南芥UFD1类似蛋白有74％的同源性。在多肽链的N-端具有在真核生物中高度保守的UFD1结构域。我们将该基因定位在小麦的第六染色体群并将其命名为了UFD1。Southern杂交和数据库搜索表明植物的UFD1基因是单拷贝或低拷贝的。无论是在单子叶中还是在双子叶植物中,UFD1蛋白都高度同源。除了N端UFD1结构域外,该类蛋白还有3个高度保守的C端结构域。TUFD1基因在小麦幼苗的根、茎、胚芽鞘、叶片以及幼穗和腊熟期子粒中呈组成性表达。     

小麦泛素融合降解蛋白基因的克隆及特征分析

酵母UFD1基因编码的泛素融合降解蛋白是泛素依赖性降解系统或泛素融合降解途径中的一个关键因子.利用RT-PCR技术在小麦(Triticum aestivum L.)中分离到一个UFD1类似基因.该基因的编码区长948 bp,编码长315个氨基酸的多肽,其氨基酸序列与GenBank中登录的一个拟南芥UFD1类似蛋白有74%的同源性.在多肽链的N-端具有在真核生物中高度保守的UFD1结构域.我们将该基因定位在小麦的第六染色体群并将其命名为TUFD1.South-ern杂交和数据库搜索表明植物的UFD1基因是单拷贝或低拷贝的.无论是在单子叶中还是在双子叶植物中,UFD1蛋白都高度同源.除了N端UFD1结构域外,该类蛋白还有3个高度保守的C端结构域.TUFD1基因在小麦幼苗的根、茎、胚芽鞘、叶片以及幼穗和腊熟期子粒中呈组成性表达.     

花同源异型MADS-box 基因在被子植物中的功能保守性和多样性

MADS-box基因家族成员作为转录调控因子在被子植物花发育调控中发挥关键作用。本文以模式植物拟南芥(Arabidopsis thaliana) 和水稻 (Oryza sativa)为例, 综述了近10年来对被子植物(又称有花植物)两大主要类群——核心真 双子叶植物和单子叶植物花同源异型MADS-box基因的研究成果, 分析MADS-box基因在被子植物中的功能保守性和多样性,同时探讨双子叶植物花发育的ABCDE模型在多大程度上适用于单子叶植物。     

农杆菌介导单子叶植物基因转化研究进展

农杆菌介导基因转化系统是双子叶植物基因转化的普通而有效的手段,其优点倍受重视,近年来又广泛用于曾被认为不在农杆菌宿主范围之内的单子叶植物的基因转化研究,并在很多重要粮食作物上获得成功,例如水稻、玉米、大麦、小麦等。本文就农杆菌转化的优点,转化机理以及对单子叶植物转化的研究进展作一概述。     

农杆菌介导单子叶植物基因转化研究进展

农杆菌介导基因转化系统是双子叶植物基因转化的普通而有效的手段, 其优点倍受重视,近年来又广泛用于曾被认为不在农杆菌宿主范围之内的单子叶植物的基因转化研究,并在很多重要粮食作物上获得成功,例如水稻、玉米、大麦、小麦等。本文就农杆菌转化的优点,转化机理以及对单子叶植物转化的研究进展作一概述     

花同源异型MADS-box基因在被子植物中的功能保守性和多样性

MADS-box基因家族成员作为转录调控因子在被子植物花发育调控中发挥关键作用。本文以模式植物拟南芥（Arabidopsis thaliana）和水稻（Oryza sativa）为例,综述了近10年来对被子植物（又称有花植物）两大主要类群——核心真双子叶植物和单子叶植物花同源异型MADS-box基因的研究成果,分析MADS-box基因在被子植物中的功能保守性和多样性,同时探讨双子叶植物花发育的ABCDE模型在多大程度上适用于单子叶植物。     

籽粒苋丙酮酸磷酸二激酶(PPDK)基因的密码子偏好性

运用CHIPS、CUSP和CodonW等程序分析了双子叶C₄植物籽粒苋(Amaranthus hypochondriacus)丙酮酸磷酸二激酶(PPDK)基因的密码子偏好性, 并与马铃薯(Solanum tuberosum)和苜蓿(Medicago truncatula)等双子叶植物及水稻(Oryza sativa)和玉米(Zea mays)等单子叶植物进行了比较, 建立了聚类树状图, 以期在作物高光效基因工程中为籽粒苋PPDK基因选择合适的受体植物提供依据。研究结果表明, 籽粒苋PPDK基因偏好于以A或T结尾的密码子, 与其它几种被比较的双子叶作物的PPDK基因密码子偏好性趋势一致, 而玉米和水稻等单子叶植物更偏好使用以G或C结尾的密码子。PPDK基因密码子使用偏好性的系统聚类分析表明, 籽粒苋与马铃薯和苜蓿等双子叶植物聚为一类, 而稗草(Echinochloa crusgalli)、玉米和高粱(Sorghum bicolor)等单子叶植物聚为一类, 与系统进化地位一致。但单子叶植物水稻的密码子偏好性与籽粒苋较为接近, 与玉米和高粱相差较远。为了选择合适的蛋白质表达系统, 比较并分析了籽粒苋PPDK基因的密码子偏好性与大肠杆菌(Escherichia coli)及酵母菌的异同, 发现其与酵母菌的差异小于大肠杆菌, 表明选择酵母菌表达系统更为合适。     

利用烟草瞬时表达系统检测高等植物基因的剪接加工

解析基因的剪接加工机制是了解植物形态建成、生长发育和逆境胁迫应答的重要环节．与动物相比,植物中相应的研究进展较为缓慢．利用农杆菌介导的烟草瞬时表达系统,分别对单子叶植物水稻BADH2和双子叶植物拟南芥GR7基因片段在烟草叶片中的转录后剪接加工进行分析．结果表明,一些重要剪接调控元件在植物中保守存在,而烟草瞬时表达系统可以作为研究高等植物剪接调控的重要工具,快捷灵敏地检测基因的剪接加工方式．     

丹参miR408基因前体序列的克隆及表达分析

MiR408是植物中一类高度保守的miRNA,其靶基因编码含铜蛋白,miR408的表达受植物生长发育和环境条件的显著影响。拟南芥中miR408在HY5-SPL7基因网络中起着关键作用,而HY5-SPL7基因网络可介导拟南芥对光照和铜离子的协调应答,进一步表明miR408在植物对环境的响应中发挥了核心作用。本研究基于丹参基因组Survey数据库,从中搜索并克隆得到366 bp的含有稳定茎环结构的miR408前体序列及其上游723 bp片段的启动子区,Gen Bank登录号分别为KU360384和KU360385,并将miR408的前体序列命名为SmMIR408。生物信息学分析结果显示:Sm-MIR408上游启动子序列与模式植物拟南芥Ath-MIR408、水稻OsaMIR408启动子区具有许多相同的顺式作用元件,如G-box、CGTCA-motif、TGACG-motif、GTAC-motif等,而HSE、CATT-motif作用元件仅存在于丹参启动子区;miR408成熟序列在不同物种中具有很高的保守性;基于不同物种miR408前体序列构建的系统进化树显示,来源于单子叶植物的miR408前体序列聚为一支,来源于双子叶植物的miR408前体序列聚为另一支。实时定量PCR分析结果显示:Sm-MIR408在丹参的根、茎、叶、花中都有表达,其在花中的表达量最高,根中最低;Sm-MIR408的表达受茉莉酸甲酯和伤害的抑制,黑暗和持续光照也可抑制该基因的表达。Sm-MIR408基因的克隆与表达分析为今后研究丹参miR408的功能奠定了基础。     

植物气孔发育机制研究进展

气孔是分布在植物表面的微孔, 是植物水分散失和与外界环境进行气体交换的门户。经过多年的研究, 气孔发育过程中重要调节因子陆续被发现。气孔复合体结构、发育起始模式以及在双子叶植物和单子叶植物中的分布都有较大差异。该文综述了双子叶植物拟南芥(Arabidopsis thaliana)气孔发育过程中调控因子、细胞极性分裂以及环境因子和植物激素调控气孔发育的机制; 还阐述了单子叶植物玉米(Zea mays)、二穗短柄草(Brachypodium distachyum)和水稻(Oryza sativa)气孔发育方面的研究进展, 并展望了气孔发育的研究方向。     

Maize U2 snRNAs: gene sequence and expression.

The complexity of plant U-type small nuclear ribonucleoprotein particles (UsnRNPs) may represent one level at which differences in splicing between animals and plants and between monocotyledonous and dicotyledonous plants could be effected. The maize (monocot.) U2snRNA multigene family consists of some 25 to 40 genes which from RNA blot and RNase protection analyses produce U2snRNAs varying in both size and sequence. The first 77 nucleotides of the maize U2-27 snRNA gene are identical to U2snRNA genes of Arabidopsis (dicot). Despite much lower sequence homology in the remaining 120 nucleotides the secondary structure of the RNA is conserved. The difference in splicing between monocot. and dicot. plants cannot be explained on the basis of sequence differences between monocot, and dicot. U2snRNAs in the region which may interact with intron branch point sequences.     

Molecular analysis of dicot and monocot small nuclear RNA populations.

Oligonucleotides directed against conserved small nuclear RNA (snRNA) sequences have been used to identify the individual U1, U2, U4, U5, and U6 snRNAs in dicot and monocot nuclei. The plant snRNA populations are significantly more heterogeneous than the mammalian or Saccharomyces cerevisiae snRNA populations. U6 snRNA exists as a single species of similar size in monocot and dicot nuclei. The abundance and molecular weights of the U1, U2, U4, and U5 snRNAs expressed in monocot and dicot nuclei are significantly different. Whereas most dicot nuclei contain one or two predominant forms of U2 snRNA and a small number of U4 snRNAs, monocot nuclei contain multiple forms of U2 snRNA ranging from 208 to 260 nucleotides and multiple forms of U4 snRNA from 159 to 176 nucleotides. Multiple forms of U1 and U5 snRNA exist in both plant groups. All prominent size variants of U1, U2, U4, and U5 snRNA identified in monocot nuclei can be immunoprecipitated with anti-trimethylguanosine antibody. We conclude that the sizes and number of snRNA molecules involved in intron excision differ considerably in dicot and monocot nuclei. In wheat nuclei, we have identified an additional U1-like RNA that is differentially expressed during development.     

Molecular comparison of monocot and dicot U1 and U2 snRNAs

To elucidate differences between the pre-mRNA splicing components in monocots and dicots, we have cloned and characterized several U1 and U2 snRNA sequence variants expressed in wheat seedling nuclei. Primer extension sequencing on wheat and pea snRNA populations has demonstrated that two 5'-terminal nucleotides found in most other U1 snRNAs are missing/modified in many plant U1 snRNAs. Comparison of the wheat U1 and U2 snRNA variants with their counterparts expressed in pea nuclei has defined regions of structural divergence between monocot and dicot U1 and U2 snRNAs. The U1 and U2 snRNA sequences involved in RNA:RNA interaction with pre-mRNAs are absolutely conserved. Significant differences occur between wheat and pea U1 snRNAs in stem I and II structures implicated in the binding of U1-specific proteins suggesting that the monocot and dicot U1-specific snRNP proteins differ in their binding specificities. Stem III structures, which are required in mammalian systems for splicing complex formation but not for U1-specific protein binding, differ more extensively than stems I, II, or IV. In U2 snRNAs, the sequence differences between these two species are primarily localized in stem III and in stem IV which has been implicated in snRNP protein binding. These differences suggest that monocot and dicot U1 and U2 snRNPs represent distinct entities that may have monocot- and dicot-specific snRNP protein variants associated with each snRNA.     

Conserved sequences in the U2 snRNA-encoding genes of Kinetoplastida do not include the putative branchpoint recognition region

The U2 small nuclear RNA (snRNA) of Trypanosoma brucei gambiense, a flagellated protozoon of the order Kinetoplastida, is 148 nucleotides (nt) long, and thus the smallest U2 snRNA identified so far. To examine the evolutionary conservation of this RNA among Kinetoplastida, we have cloned and sequenced the U2 genes from Trypanosoma congolense and Leishmania mexicana amazonensis, which are 145 and 141 nt in length, respectively. The sequences of the Kinetoplastida U2 snRNAs are essentially identical in the 5' half of the molecule. Surprisingly, the putative branch site recognition sequence of L. m. amazonensis U2 snRNA shows two nt changes when compared with the other two U2 snRNAs. The sequence of the 3' half of the Kinetoplastida U2 snRNAs is less conserved with T. congolense and L. m. amazonensis RNAs showing 23 and 35 nt sequence variations, respectively, when compared with the corresponding sequence of the T. b. gambiense U2 snRNA. Alignment of the flanking regions of the U2 genes revealed several elements which are conserved both in sequence and in position relative to the U2 coding region and which may function in the biosynthesis of U2 snRNAs. One upstream element specifically binds protein factor(s) present in T. brucei nuclear extracts.     

cDNA cloning of U1, U2, U4 and U5 snRNA families expressed in pea nuclei.

Differences observed between plant and animal pre-mRNA splicing may be the result of primary or secondary structure differences in small nuclear RNAs (snRNAs). A cDNA library of pea snRNAs was constructed from anti-trimethylguanosine (m3(2,2,7)G immunoprecipitated pea nuclear RNA. The cDNA library was screened using oligo-deoxyribonucleotide probes specific for the U1, U2, U4 and U5 snRNAs. cDNA clones representing U1, U2, U4 and U5 snRNAs expressed in seedling tissue have been isolated and sequenced. Comparison of the pea snRNA variants with other organisms suggest that functionally important primary sequences are conserved phylogenetically even though the overall sequences have diverged substantially. Structural variations in U1 snRNA occur in regions required for U1-specific protein binding. In light of this sequence analysis, it is clear that the dicot snRNA variants do not differ in sequences implicated in RNA:RNA interactions with pre-mRNA. Instead, sequence differences occur in regions implicated in the binding of small ribonucleoproteins (snRNPs) to snRNAs and may result in the formation of unique snRNP particles.     

水稻重复序列RRD3:植物启动子及其在RNA干涉中的应用

RRD3是通过复性动力学从水稻基因组中克隆到的一个中度重复序列,序列分析揭示其含有多个保守的启动子元件,包括4个TATA-boxes和CAAT-box,在Escherichia coli及哺乳动物表达系统中均表现出启动子活性。我们将RRD3片段插入到植物启动子捕获载体pCAMBIA1391Z中,检测RRD3片段是否在植物中也有启动子活性,转基因烟草再生植株和水稻愈伤组织均显示了gusA基因的表达,表明其在单子叶和双子叶植物中均可行使启动子功能。基于RRD3的双效启动子特性,我们设计并构建了植物通用双元RNAi载体pCRiRRD3,适于进行植物的RNA干涉实验研究。我们在植物RNAi载体pCRiRRD3的内含子(200bp)的上下游分别引入了2个多克隆位点,方便了正义及反义片段的接入。转基因烟草植株的GUS组织化学及荧光定量分析表明该载体可有效进行RNA沉默。这些研究结果表明,利用单子叶和双子叶双效活性的RRD3启动子而构建的植物RNAi载体,可同时有效地应用于大部分单子叶和双子叶植物的RNAi实验及研究。     

Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNa polymerase III

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to -amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.     

Evidence for the presence of a small U5-like RNA in active trans-spliceosomes of Trypanosoma brucei.

The existence of the Trypanosoma brucei 5' splice site on a small RNA of uniform sequence (the spliced leader or SL RNA) has allowed us to characterize the RNAs with which it interacts in vivo by psoralen crosslinking treatment. Analysis of the most abundant crosslinks formed by the SL RNA allowed us previously to identify the spliced leader-associated (SLA) RNA. The role of this RNA in trans-splicing, as well as the possible existence of an analogous RNA interaction in cis-splicing, is unknown. We show here that the 5' splice site region of the SL RNA is also crosslinked in vivo to a second small RNA. Although it is very small and lacks a 5' trimethylguanosine (TMG) cap, the SLA2RNA possesses counterparts of the conserved U5 snRNA stem-loop 1 and internal loop 1 sequence elements, as well as a potential trypanosome snRNA core protein binding site; these combined features meet the phylogenetic definition of U5 snRNA. Like U5, the SLA2 RNA forms an RNP complex with the U4 and U6 RNAs, and interacts with the 5' splice site region via its putative loop 1 sequence. In a final analogy with U5, the SLA2 RNA is found crosslinked to a molecule identical to the free 5' exon splicing intermediate. These data present a compelling case for the SLA2 RNA not only as an active trans-spliceosomal component, but also for its identification as the trypanosome U5 structural homolog. The presence of a U5-like RNA in this ancient eukaryote establishes the universality of the spliceosomal RNA core components.