基于孢子形态和分子证据探讨鳞盖蕨属(碗蕨科)系统分类

孢粉学是解决植物分类中疑难类群物种微形态分化的重要方法, 随着分子系统学的发展, 结合这两门学科的优势可以更加有效地解决疑难类群的分类学问题。鳞盖蕨属(Microlepia)是一个分类困难的疑难类群, 采用孢粉学与分子系统学一一对应的方法, 以及居群取样方式, 选取280份样本, 联合4个叶绿体片段(rbcL、trnL-F、psbA-trnH和rps4), 采用最大似然法和贝叶斯法构建该属的系统发生关系, 在此基础上对凭证标本中100份材料的孢子进行观察和分析。综合分子系统学和孢粉学的研究结果, 得出结论: (1) 在形态学研究中广泛被接受的15个物种得到了单系支持, 并厘清了分类困难的复合群; (2) 发现边缘鳞盖蕨(M. marginata)可能存在隐性种; (3) 建议恢复过去归并处理为异名的瑶山鳞盖蕨(M. yaoshanica)、罗浮鳞盖蕨(M. lofoushanensis)、四川鳞盖蕨(M. szechuanica)以及滇西鳞盖蕨(M. subspeluncae); (4) 提出鳞盖蕨属可能存在杂交现象; (5) 提出鳞盖蕨属完整的属下分类建议。     

薄鳞蕨属的分类研究

在野外考察和室内标本研究的基础上,对薄鳞蕨属（中国蕨科）的分类进行了研究。 将2种 (华西薄鳞蕨和察隅薄鳞蕨) 和2变种（大叶薄鳞蕨和宽叶薄鳞蕨）进行了归并处理, 承认该属有4种1变种。     

叉蕨科4属5种植物配子体的发育模式及其系统学意义

利用光学显微镜详细观察了叉蕨科（Aspidiaceae）4属5种植物,即肋毛蕨属（Ctenitis（C．Chr．）C．Chr．）的亮鳞肋毛蕨（C．subglandulosa（Hance）Ching）和海南肋毛蕨（C．decurrenti-pmnata（Ching）Ching）、轴脉蕨属（Ctenitopsis Ching ex Tard-Blot et C．Chr．）的轴脉蕨（C．sagenioides（Mett．）Ching）、黄腺羽蕨属（Pleocnemia Presl）的黄腺羽蕨（P．winitti Holtt．）以及叉蕨属（Tectaria Cav．）的剑叶叉蕨（T.leptophylla（C．H．Wright）Ching）的配子体发育过程,记录了配子体各发育阶段的模式特征,认为这5种植物的孢子、丝状体、片状体、生长点、翼片、细胞、毛状体和假根等具有稳定的系统学意义。检索结果与该科的经典分类结果基本相似,并在此基础上编写了各分类群的检索表。本研究为叉蕨科系统学研究积累了详实的配子体形态学资料。     

国产18种鳞盖蕨属植物叶表皮微形态特征及其系统学意义

鳞盖蕨属是一个自然类群,由于各类群之间的形态差异以及种内变异比较大,因此其分类一直存在争议。该研究利用光学显微镜(LM)和扫描电子显微镜(SEM)对鳞盖蕨属18种植物(含3变种)的叶表皮微形态进行了观察和比较,并结合形态学、孢粉学和分子系统学探讨其系统学意义。结果表明:在光学显微镜下,18种鳞盖蕨属植物叶上、下表皮的脉上均有毛,叶表皮细胞都为不规则形,垂周壁为深波状或波状; 气孔都分布在下表皮,气孔器类型主要有极细胞型和腋下细胞型两种,叶表皮微形态特征支持鳞盖蕨属是碗蕨科中的一个单系类群; 叶上、下表皮脉间毛的变异特征支持光叶鳞盖蕨、毛叶边缘鳞盖蕨和二回边缘鳞盖蕨处理为边缘鳞盖蕨变种; 在扫描电镜下,叶上表皮角质层多为脊状凸起,多数还具丝状纹饰,角质膜特征与叶片回数有一定的相关性; 共环极细胞型、聚腋下细胞型、不等细胞型和不规则四细胞型只在少数种出现,气孔外拱盖多数凹陷,光滑或有颗粒; 气孔外拱盖内缘平滑、浅波状或齿状; 保卫细胞两极大多数有“T”型加厚,不同种间叶表皮微形态特征表现出一定差异。该研究结果为其分类、演化、系统位置等进一步研究提供了参考。     

论拟贯众属的系统位置

综合形态学、叶表皮解剖学、孢粉学、分子系统学及生物地理学的证据,认为拟贯众属被置于鳞毛蕨科的处理是错误的。多方面证据支持这个属与叉蕨科的一些属有着较近的亲缘关系,我们建议将拟贯众属从鳞毛蕨科中移出而暂时置于叉蕨科内。     

线蕨属（水龙骨科）的系统发育研究：来自叶绿体rbcL和rps4-trnS序列的证据

线蕨属Colysis 植物主要分布于亚洲热带和亚热带地区,少数种类分布至非洲、澳大利亚(昆士兰)及新几内亚地区.自1849年成立以来,线蕨属的分类范畴和系统位置一直有待确定.本文利用叶绿体基因组的rbcL、rps4基因和rps4-trnS基凶间隔区序列,运用最大简约法和贝叶斯方法分析了线蕨属及其近缘类群的系统演化关系.研究结果显示:(1)线蕨属和薄唇蕨属Leptochilus(含似薄唇蕨属Paraleptochilus)组成一个支持率很高的单系分支(C-L Clade),但是薄唇蕨属的成员位于线蕨属的不同支系内,支持线蕨属和薄唇蕨属合并为一个属;(2)瘤蕨属Phymatosorus单独形成一个单系分支;(3)星蕨属Microsorum是一个多系类群,除Microsorium linguiforme、M.varians和M.pustulatum与马来群岛的Lecanopteris聚在一起外,其他的星蕨属成员均位于不同的支系上.本文的系统发育分析结果为线蕨属和薄唇蕨属的分类处理提供了分子系统学的证据.     

用叶绿体rbcL和rps4-trnS区序列确定亚洲特有单型属——篦齿蕨属的系统位置

篦齿蕨属Metapolypodium Ching是亚洲大陆特有植物, 仅含篦齿蕨Metapolypodium manmeiense (Christ) Ching 1种, 分布于中国西南及其邻近地区, 其系统位置一直是一个有待解决的问题。本文用叶绿体rbcL和rps4-trnS区序列探讨篦齿蕨属的系统位置。我们对篦齿蕨及其近缘类群的叶绿体rbcL和rps4-trnS区进行PCR扩增和序列测定。用最大简约法、邻接法和贝叶斯推断法对自测序列结合由GenBank下载的rbcL和rps4-trnS区序列进行独立和联合的系统发育分析, 所构建的系统发生树的拓扑结构基本一致。结果显示: 篦齿蕨属与多足蕨属Polypodium L. emend. Ching的关系较疏远, 而与水龙骨属Polypodiodes Ching和拟水龙骨属Polypodiastrum Ching的成员聚成一个支系, 与栗柄水龙骨Polypodiodes microrhizoma (C. B. Clarke ex Baker) Ching的关系最近。根据本文的研究结果, 我们赞成秦仁昌1978年的分类系统, 即分别承认多足蕨属、篦齿蕨属、水龙骨属、拟水龙骨属和棱脉蕨属Schellolepis J. Sm.为独立的类群。根据分子系统学证据, 宜将栗柄水龙骨转移到篦齿蕨属中。     

宽带蕨属（水龙骨科）的系统学研究

对喜马拉雅和横断山地区特有的水龙骨科的宽带蕨属Platygyria Ching&S.K.Wu植物进行了形态学、分类学、生态和植物地理学的观察与分析,探讨了该属植物的系统位置、亲缘关系和生态适应性,推测宽带蕨属是随着喜马拉雅山脉的隆升,由瓦韦属Lepisorus（J.Sm.）Ching网眼瓦韦群植物适应高寒生态环境演化而来的新生类群。确定宽带蕨属有5种,即川西宽带蕨P.soulieana（Christ）X.C.Zhang&Q.R.Liu、多变宽带蕨P.variabilis Ching&S.K.Wu、耳基宽带     

薄鳞蕨属(中国蕨科)植物的孢子形态研究

对薄鳞蕨属5种3变种的孢子形态进行了光镜和扫描电镜观察。该属植物的孢子明显地分为两种类型：薄叶薄鳞蕨和杜氏薄鳞蕨的孢子为近球形,周壁为鸡冠状或拟网状纹饰;而华北薄鳞蕨、华西薄鳞蕨和绒毛薄鳞蕨的孢子为四面体状球形,周壁表面近光滑。第一种类型的孢子也存在于粉背蕨属,因此薄鳞蕨属和粉背蕨属的关系仍需进一步的研究。     

金粉蕨属的分子系统学研究——基于5种叶绿体DNA序列片段

金粉蕨属（Onychium Kaulfuss）隶属广义凤尾蕨科中的凤尾蕨亚科。迄今为止,该属属下分组及种间界定等仍有诸多问题亟待解决。本研究选取5个叶绿体DNA 序列片段 （rbcL/atpA/matK/trnL-trnF/trnG-trnR）,采用最大似然法（ML）和贝叶斯法（BI）构建金粉蕨属的系统发育树。结果表明：（1）金粉蕨属的9个成员被分置于两大支上。其中野雉尾金粉蕨（Onychium japonicum(Thunberg) Kunze）、西藏金粉蕨（O.tibeticum Ching & S.K.Wu）、木坪金粉蕨（O.moupinense Ching）、湖北金粉蕨（O.moupinense var. ipii(Ching) K.H.Shing）、栗柄金粉蕨（O.japonicum var. lucidum(D.Don) Christ）、黑足金粉蕨（O.cryptogrammoides Christ）、繁羽金粉蕨（O.plumosum Ching）聚为一支;而金粉蕨（O.siliculosum(Desvaux) C.Christensen）和蚀盖金粉蕨（O.tenuifrons Ching）则聚为另一支,可为该属的属下分组提供分子系统学证据;（2）野雉尾金粉蕨与栗柄金粉蕨在系统树中并没有聚在一起,而是被其它类群分割开来,不支持将后者作为野雉尾金粉蕨的变种,建议将栗柄金粉蕨提升为种的等级;（3）系统树上木坪金粉蕨与湖北金粉蕨的样本聚在一个细支上,支持《中国植物志》将湖北金粉蕨作为木坪金粉蕨变种的分类处理;（4）西藏金粉蕨与野雉尾金粉蕨聚在一起,并得到较高的支持,说明两者的关系近缘。     

DNA barcoding of Orchidaceae in Korea

Species of Orchidaceae are under severe threat of extinction mainly due to overcollection and habitat destruction; accurate identification of orchid species is critical in conservation biology and sustainable utilization of orchids as plant resources. We examined 647 sequences of the cpDNA regions rbcL, matK, atpF‐atpH IGS, psbK‐psbI IGS and trnH‐psbA IGS from 89 orchid species (95 taxa) and four outgroup taxa to develop an efficient DNA barcode for Orchidaceae in Korea. The five cpDNA barcode regions were successfully amplified and sequenced for all chlorophyllous taxa, but the amplification and sequencing of the same regions in achlorophyllous taxa produced variable results. psbK‐psbI IGS showed the highest mean interspecific K2P distance (0.1192), followed by matK (0.0803), atpF‐atpH IGS (0.0648), trnH‐psbA IGS (0.0460) and rbcL (0.0248). The degree of species resolution for individual barcode regions ranged from 60.5% (rbcL) to 83.5% (trnH‐psbA IGS). The degree of species resolution was significantly enhanced in multiregion combinations of the five barcode regions. Of the 26 possible combinations of the five regions, six provided the highest degree of species resolution (98.8%). Among these, a combination of atpF‐atpH IGS, psbK‐psbI IGS and trnH‐psbA IGS, which comprises the least number of DNA regions, is the best option for barcoding of the Korean orchid species.     

A Local Role for the Small Ribosomal Subunit Primary Binder rpS5 in Final 18S rRNA Processing in Yeast

In vivo depletion of the yeast small ribosomal subunit (SSU) protein S5 (rpS5) leads to nuclear degradation of nascent SSUs and to a perturbed global assembly state of the SSU head domain. Here, we report that rpS5 plays an additional local role at the head/platform interface in efficient SSU maturation. We find that yeast small ribosomal subunits which incorporated an rpS5 variant lacking the seven C-terminal amino acids have a largely assembled head domain and are exported to the cytoplasm. On the other hand, 3′ processing of 18S rRNA precursors is inhibited in these ribosomal particles, although they associate with the putative endonuclease Nob1p and other late acting 40S biogenesis factors. We suggest that the SSU head component rpS5 and platform components as rpS14 are crucial constituents of a highly defined spatial arrangement in the head – platform interface of nascent SSUs, which is required for efficient processing of the therein predicted SSU rRNA 3′ end. Positioning of rpS5 in nascent SSUs, including its relative orientation towards platform components in the head-platform cleft, will depend on the general assembly and folding state of the head domain. Therefore, the suggested model can explain 18S precursor rRNA 3′ processing phenotypes observed in many eukaryotic SSU head assembly mutants.     

扇蕨配子体发育与濒危机制的探讨

采用改良Knop’s固体培养基、原生境腐殖质土和红壤分别培养扇蕨（Neocheiropteris palmatopedate）孢子,光学显微镜及解剖镜下观察记录其孢子萌发及配子体发育过程,比较了3种培养方法对其配子体发育和有性繁殖的影响,并在此基础上探讨了扇蕨的濒危原因。结果表明：成熟孢子黄褐色,赤道面观为豆形,极面观椭圆形,单裂缝。孢子萌发类型为书带蕨型,原叶体发育为槲蕨型。成熟原叶体呈心脏形。毛状体在原叶体阶段出现。有性生殖周期长及配子体发育成幼孢子体的百分率低是扇蕨在配子体世代的主要濒危原因。此外,红壤固有的理化性质导致扇蕨配子体发育极其缓慢、精子器和颈卵器发生的时间间隔过长使其不能受精产生孢子体。原生植被遭受破坏引起的林下腐殖质土消失、红壤裸露,加剧了扇蕨的濒危。     

Regulation of ribosomal protein S6 phosphorylation by casein kinase 1 and protein phosphatase 1

Ribosomal protein S6 (rpS6) is a critical component of the 40 S ribosomal subunit that mediates translation initiation at the 5'-m(7)GpppG cap of mRNA. In response to mitogenic stimuli, rpS6 undergoes ordered C-terminal phosphorylation by p70 S6 kinases and p90 ribosomal S6 kinases on four conserved Ser residues (Ser-235, Ser-236, Ser-240, and Ser-244) whose modification potentiates rpS6 cap binding activity. A fifth site, Ser-247, is also known to be phosphorylated, but its function and regulation are not well characterized. In this study, we employed phospho-specific antibodies to show that Ser-247 is a target of the casein kinase 1 (CK1) family of protein kinases. CK1-dependent phosphorylation of Ser-247 was induced by mitogenic stimuli and required prior phosphorylation of upstream S6 kinase/ribosomal S6 kinase residues. CK1-mediated phosphorylation of Ser-247 also enhanced the phosphorylation of upstream sites, which implies that bidirectional synergy between C-terminal phospho-residues is required to sustain rpS6 phosphorylation. Consistent with this idea, CK1-dependent phosphorylation of rpS6 promotes its association with the mRNA cap-binding complex in vitro. Additionally, we show that protein phosphatase 1 (PP1) antagonizes rpS6 C terminus phosphorylation and cap binding in intact cells. These findings further our understanding of rpS6 phospho-regulation and define a direct link between CK1 and translation initiation.     

Accuracy modulating mutations of the ribosomal protein S4-S5 interface do not necessarily destabilize the rps4-rps5 protein–protein interaction

During the process of translation, an aminoacyl tRNA is selected in the A site of the decoding center of the small subunit based on the correct codon–anticodon base pairing. Though selection is usually accurate, mutations in the ribosomal RNA and proteins and the presence of some antibiotics like streptomycin alter translational accuracy. Recent crystallographic structures of the ribosome suggest that cognate tRNAs induce a “closed conformation” of the small subunit that stabilizes the codon–anticodon interactions at the A site. During formation of the closed conformation, the protein interface between rpS4 and rpS5 is broken while new contacts form with rpS12. Mutations in rpS12 confer streptomycin resistance or dependence and show a hyperaccurate phenotype. Mutations reversing streptomycin dependence affect rpS4 and rpS5. The canonical rpS4 and rpS5 streptomycin independent mutations increase translational errors and were called ribosomal ambiguity mutations (ram). The mutations in these proteins are proposed to affect formation of the closed complex by breaking the rpS4-rpS5 interface, which reduces the cost of domain closure and thus increases translational errors. We used a yeast two-hybrid system to study the interactions between the small subunit ribosomal proteins rpS4 and rpS5 and to test the effect of ram mutations on the stability of the interface. We found no correlation between ram phenotype and disruption of the interface.     

The regulation of hepatic protein synthesis during fasting in the rat

We have studied translational control in the model of 48 h of fasting in the rat. Our initial observations showed a paradoxical increase in ribosomal protein S6 (rpS6) phosphorylation and a decrease in eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation. These effects, which would favor an increase in protein synthesis, could be attributed to increased circulating concentrations of branched-chain amino acids in fasting. To determine what mechanisms might account for decreased hepatic translation in fasting, we examined the cap binding complex. eIF4E-bound 4E-BP1 did not increase. However, eIF4E-bound eIF4G and total cellular eIF4G were profoundly decreased in fasted liver. eIF4G mRNA levels were not lower after fasting. Based on the hypothesis that decreased eIF4G translation might account for the reduced eIF4G content, we fractionated ribosomes by sucrose density centrifugation. Immunoblotting for rpS6 showed modest polysomal disaggregation upon fasting. PCR analysis of polysome profiles revealed that a spectrum of mRNAs undergo different translational regulation in the fasted state. In particular, eIF4G was minimally affected by fasting. This indicated that reduced eIF4G abundance in fasting may be a function of its stability, whereas its recovery upon refeeding is necessarily independent of its own involvement in the cap binding complex. Western immunoblotting of polysome fractions showed that phosphorylated rpS6 was disproportionately present in translating polysomes in fed and fasted animals, consistent with a role in translational control. However, the translation of rpS8, an mRNA with a 5'-oligopyrimidine tract, did not coincide with rpS6 phosphorylation, thus dissociating rpS6 phosphorylation from the translational control of this subset of mRNAs.     

Human ribosomal protein S13 inhibits splicing of its own pre-mRNA

Recombinant human ribosomal protein (rp) S13 was shown to specifically bind with its own pre-mRNA fragment containing the first exon, first intron, second exon, and a part of the second intron and to inhibit its splicing in vitro. The binding of rpS13 was specific: recombinant human rpS10 and rpS16 bound with the fragment to a lower extent. Moreover, rpS13 binding with the rpS13 pre-mRNA fragment was inhibited by non-labeled poly(AU) and an adenovirus pre-mRNA fragment to a lower extent than by the nonlabeled rpS13 pre-mRNA fragment. The specificity of splicing inhibition was inferred from the finding that, in contrast to rpS13, recombinant rpS10 and rpS16 did not affect the efficiency of first intron excision from the rpS13 pre-mRNA fragment. Enzymatic footprinting was used to determine the rpS13 pre-mRNA nucleotides whose accessibility to RNases T1, T2, and V1 changed in the presence of rpS13. Such nucleotides were detected close to the 5′ and 3′ splicing sites of the first intron. Analysis with the EMBOS-Align program showed that the nucleotide sequence of the first intron of the mammalian rpS13 pre-mRNA is conserved to a greater extent as compared with the other introns. It was assumed that the first intron plays an important role in regulating the expression of the rpS13 gene at the splicing level in all mammals.     

Human ribosomal protein S16 inhibits excision of the first intron from its own pre-mRNA

Recombinant human ribosomal protein S16 (rpS16) is shown to bind specifically to a fragment of its own pre-mRNA that includes exons 1 and 2, intron 1, and part of intron 2, and to inhibit the splicing of the fragment in vitro. The weaker binding of other recombinant human ribosomal proteins, S10 and S13, to this pre-mRNA fragment indicated that the binding of rpS16 was specific. Besides, the poly(AU) and rpS16 mRNA fragment insignificantly affected the binding of rpS16 to its pre-mRNA, providing another evidence that the interaction was specific. rpS16 specifically inhibited the splicing of the pre-mRNA fragment, whereas recombinant rpS10 and rpS13 did not affect intron excision from this pre-mRNA fragment in contrast to rpS16. Those positions in rpS16 pre-mRNA fragment that were protected by rpS16 from cleavage by RNases T1, T2, and V1 were found to be located closely to the branch point and 3’ splice site in the pre-mRNA. The obtained results suggest the possibility of the autoregulation of rpS13 pre-mRNA splicing through the feedback mechanism.     

PRMT3 inhibits ubiquitination of ribosomal protein S2 and together forms an active enzyme complex

Protein arginine methyltransferase 3 (PRMT3) comprises a region not required for catalytic activity in its amino-terminus and the core domain catalyzing protein arginine methylation. PRMT3 has been shown to interact with the 40S ribosomal protein S2 (rpS2) and methylate arginine residues in the arginine-glycine (RG) repeat region in the amino-terminus of rpS2. We investigated the biological implications of this interaction by delineating the domains that mediate binding between PRMT3 and rpS2. The rpS2 (100-293 amino acids) domain, but not the amino-terminus of rpS2 that includes the RG repeat region was essential for binding to PRMT3 and was susceptible to degradation. The amino-terminus of PRMT3, but not its catalytic core was required for binding to and the stability of rpS2. Overexpressed rpS2 was ubiquitinated in cells, but expression of PRMT3 reduced this ubiquitination and stabilized the rpS2 protein. Recombinant PRMT3 formed an active enzyme complex with endogenous rpS2 in vitro. Recombinant rpS2 in molar excess modestly increased the enzymatic activity of PRMT3 in vitro. Our results suggest that in addition to its catalytic function, PRMT3 may control the level of rpS2 protein in cells by inhibiting ubiquitin-mediated proteolysis of rpS2, while rpS2 may regulate the enzymatic activity of PRMT3 as a likely non-catalytic subunit.