香菇B交配型位点的分子遗传学结构研究I.信息素受体和信息素前体编码基因的克隆和序列分析

本研究结合简并PCR和染色体步行两种方法研究了香菇135菌株的交配型B位点的分子遗传学结构。从135菌株的原生质体单核体1号菌株中获得了1个信息素受体编码基因LErcb1-B1和1个信息素前体编码基因LEphb1-B1。经序列比对分析,香菇的信息素受体LErcb1-B1序列与灰盖鬼伞和裂褶菌的信息素受体之间具有同源性,经SOSUI软件分析该序列具有7次跨膜结构特征。信息素前体LEphb1-B1具有CaaX基序特征。     

香菇B交配型位点的分子遗传学结构研究II.一个香菇单核体的B位点结构的分子解析

在先前的工作中,曾经运用简并PCR和染色体步行的方法从香菇中获得了1个信息素受体编码基因和1个信息素前体编码基因。根据香菇135菌株的原生质体单核体的全基因组测序信息,设计了4对引物,用于扩增香菇苏香菌株的原生质体单核体SUP2中的信息素受体编码基因STE-3的同源物及其侧翼保守基因。实验结果共获得了33,655bp的DNA序列,运用BlastX搜索对所获得的序列进行同源性分析后,发现了7个推定基因,其中有3个为信息素受体编码基因。再根据信息素前体所具有的保守基序特征,在2个信息素受体编码基因附近发现了4个信息素前体编码基因。首次对香菇的B交配型位点的分子遗传学结构有了比较全面的了解。     

水稻螟虫神经肽PBAN及其受体序列的生物信息学分析

【目的】性信息素合成激活肽(PBAN)是控制昆虫产生性信息素的激素,本文旨在分析水稻螟虫神经肽PBAN及其受体的序列。【方法】通过t Blastn同源检索从水稻螟虫基因组和转录组数据库中鉴定水稻螟虫PBAN神经肽及其受体序列,在此基础上进行序列比对及系统发生分析。【结果】发现二化螟Chilo suppressalis、三化螟Tryporyza incertulas和大螟Sesamia inferens的PBAN成熟肽序列均含有33个氨基酸残基,其C端五肽序列完全相同,3种水稻螟虫PBAN多肽相似度为54.55%~63.64%;发现二化螟PBAN受体3个异构体全长氨基酸序列(PBANR-A、PBANR-B和PBANR-C),均含有7个跨膜区域。【结论】进化树分析发现不同昆虫PBAN神经肽及其受体存在一定的保守性和多样性,并且在进化树上的位置几乎与昆虫系统发育分类一致,推测PBAN神经肽和PBAN受体在昆虫系统进化过程中可能存在协同进化现象。本研究为水稻螟虫PBAN神经肽及其受体的结构和功能分析提供基础。     

基于线粒体DNA控制区序列变异探讨黑龙江和图们江细鳞鲑属鱼类的分类地位

基于细鳞鲑属Brachymystas鱼类的线粒体DNA控制区基因序列变异,对分布于黑龙江水系中国境内的尖吻细鳞鲑(sharp-snouted lenok)和钝吻细鳞鲑(blunt-snouted lenok),及分布于图们江的图们江细鳞鲑B.tumensis进行分子系统关系研究,为进一步确定黑龙江水系2种细鳞鲑的分类学地位、有效命名及图们江细鳞鲑物种地位性提供分子生物学依据.分布于黑龙江水系的细鳞鲑种群在系统发育树中明显构成2个独立的进化分支,分别对应可经形态鉴别而差异显著的尖吻细鳞鲑和钝吻细鳞鲑,平均序列分歧为1.9%,在属内已达到种间分化水平;图们江细鳞鲑与尖吻细鳞鲑的呼玛河、乌苏里江、奎勒河等种群共同构为1个进化分支,与尖吻细鳞鲑的序列分歧(平均为1.2%)远低于与钝吻细鳞鲑的序列分歧(平均为2.2%).结合形态学的初步研究结果(图们江细鳞鲑的主要形态特征偏向于尖吻细鳞鲑),不支持图们江细鳞鲑独立种的分类地位,建议为尖吻细鳞鲑B.lenok的同物异名,也不支持普遍认为的钝吻细鳞鲑的有效学名为B.tumensis,其有效命名还待商榷,暂属未定名种.综上所述,基于基因序列分析的遗传学结果进一步验证了形态学的分类结论,即在黑龙江水系细鳞鲑属有2个独立的种,分别为尖吻细鳞鲑B.lenok和钝吻细鳞鲑B.sp.,而图们江细鳞鲑B.tumensis应归为尖吻细鳞鲑B.lenok的同物异名.     

担子菌类食用菌交配型位点结构的研究进展

大多数可栽培的食用菌是属于担子菌的大型真菌,具有复杂的交配型系统,通常涉及到两类交配型基因,即编码同源域转录因子的A交配型基因以及编码脂肽信息素和信息素受体的B交配型基因。对担子菌交配型系统的研究已经有上百年的历史,近年来随着高通量测序技术的发展,很多常见食用菌的基因组获得测序,使得我们对不同类型交配型位点的分子遗传学结构能够进行更加细致的解析。本文在概述了担子菌有性生殖系统和交配型基因分子特点的基础上,对常见食用菌中的香菇、金针菇、灵芝、糙皮侧耳、刺芹侧耳、白灵侧耳、裂褶菌、双孢蘑菇、草菇和虎皮香菇以及模式生物灰盖鬼伞等物种的交配型位点的结构进行了总结和分析。从已有的研究结果来看,常见食用菌的交配型位点的分子遗传学结构存在多样性,不同物种的交配型位点具有不同的结构特点。从物种内不同菌株之间的交配型结构比较来看,交配型基因的位置和数量也具有丰富的多样性。在分子遗传学层面对常见食用菌交配型位点结构的认识将有助于深入阐明交配型基因对子实体发育的调控以及解决食用菌生产实际中的科学问题,但是目前对食用菌交配型位点和基因的研究仍旧存在很多空白,有待于进一步深入和拓展。     

假丝酵母属疑难菌株大亚基rDNA D1/D2区域序列分析及其分类学意义*

对根据常规形态和生理生化性状难以确定分类学地位的8株假丝酵母菌,进行了以大亚基(26S) rDNA中D1/D2区域(约500~600 bp)的碱基序列分析为依据的分子分类学研究。根据系统树上所显示的供试菌株与假丝酵母属及相关子囊菌酵母已知种的亲缘关系,以及与最近缘种模式菌株D1/D2区域序列的相似性比较,确定了各个菌株的归属。本研究也显示了DNA序列分析在假丝酵母菌快速鉴定中的优越性。     

假丝酵母属疑难菌株大亚基rDNA D1/D2区域序列分析及其分类学意义

对根据常规形态和生理生化性状难以确定分类学地位的8株假丝酵母菌,进行了以大亚基(26S) rDNA中D1/D2区域(约500～600 bp)的碱基序列分析为依据的分子分类学研究.根据系统树上所显示的供试菌株与假丝酵母属及相关子囊菌酵母已知种的亲缘关系,以及与最近缘种模式菌株D1/D2区域序列的相似性比较,确定了各个菌株的归属.本研究也显示了DNA序列分析在假丝酵母菌快速鉴定中的优越性.     

假丝酵母属疑难菌株大亚基rDNAD1／D2区域序列分析及其分类学意义

对根据常规形态和生理生化性状难以确定分类学地位的8株假丝酵母菌,进行了以大亚基（26S）rDNA中D1/D2区域（均500-600bp)的碱基序列分析为依据的分子分类学研究。根据系统树上所显示的供试菌株与假丝酵母属及相关子囊菌酵母已知种的亲缘关系,以及与最近缘种模式菌株D1/D2区域序列的相似性比较,确定了各个菌株的归属,本研究也显示了DNA序列分析在假丝酵母菌快速鉴定中的优越性。     

基于28S rDNA序列的姬小蜂科(膜翅目,小蜂总科)分类

选择28S rDNA D2区基因,针对GenBank中姬小蜂科总计542条相关序列,借助Blast Align、MUSCLE及TNT等生物信息学软件进行计算分析,提出了一种基于亚科水平的姬小蜂科快速DNA分类鉴定方法。建树结果对目前分类系统中姬小蜂科4亚科分类体系(Bouek,1988)予以支持;综合分析结果基本支持对于姬小蜂亚科以及灿小蜂亚科的分族、分属方法。同时对地位不明的两属Anselmella和Ophelimus的分类学地位提出了假设。     

梧桐科分类学研究评述

梧桐科(Sterculiaceae)是锦葵目中的一个多型科,主要分布于热带和亚热带地区,只有少数种可分布到温带地区。由于该科植物的形态特征较为多样化,至今对于它的范围和所包含的属种数目在各国学者间仍没有达成共识。该文从梧桐科的分类地位和系统关系、属的分类地位和亲缘关系以及分类学新特征在梧桐科分类中的应用3个方面入手,分析了梧桐科分类学研究的历史、现状和存在的问题,特别是把来自分子资料的研究结果与传统分类进行了比较分析,试图为梧桐科的分类学研究提供更多的帮助。     

应用简并引物扩增黑木耳信息素受体基因片段

Degenerate primers, br1-F and br1-R, designed based on the conserved amino acid sequence of STE3 pheromone receptor in Schizophyllum commune, were used to amplify genomic DNA of monkaryotic parental strains(H2, J3) and fifty-nine monokaryons of their F1 progenies in Auricularia auricula. A fragment of the PCR product 811bp in length were amplified from the parental strain H2, nine monokaryons of the H2 mating –type and fifteen ones of the J3 mating –type of F1 progenies. After cloning , sequencing the fragm…     

Cloning and Analysis of Partial B Mating Gene Pheromone Receptor in Agrocybe salicacola (Strophariaceae)

A 4231bp DNA fragment of B mating type pheromone receptor from strain YAASM0711 of Agrocybe salicacola was obtained by using degenerate PCR and DNA walking techniques. The result of alignment and structure prediction of DNA sequences showed that one 1194bp nucleotides gene ASRcb1 encoding B mating pheromone receptor was found, including four introns(72bp, 49bp, 48bp and 41bp) and five extrons (217bp, 113bp, 67bp, 138bp and 449bp). The spliced open reading frame (ORF) contains 984bp nucleotides encoding 327 amino acid residues, and includes seven transmembrane protein regions as in its similar sequences of Coprinus cinerea and Laccaria bicolor pheromone receptors. The genetic evolution of pheromone receptor showed ASRcb1 was clustered with more receptors, which suggested multiple ways of evolution occurred in fungi.     

Functional characterization of an alpha-factor-like Sordaria macrospora peptide pheromone and analysis of its interaction with its cognate receptor in Saccharomyces cerevisiae

The homothallic filamentous ascomycete Sordaria macrospora possesses genes which are thought to encode two pheromone precursors and two seven-transmembrane pheromone receptors. The pheromone precursor genes are termed ppg1 and ppg2. The putative products derived from the gene sequence show structural similarity to the alpha-factor precursors and a-factor precursors of the yeast Saccharomyces cerevisiae. Likewise, sequence similarity has been found between the putative products of the pheromone receptor genes pre2 and pre1 and the S. cerevisiae Ste2p alpha-factor receptor and Ste3p a-factor receptor, respectively. To investigate whether the alpha-factor-like pheromone-receptor pair of S. macrospora is functional, a heterologous yeast assay was used. Our results show that the S. macrospora alpha-factor-like pheromone precursor PPG1 is processed into an active pheromone by yeast MATalpha cells. The S. macrospora PRE2 protein was demonstrated to be a peptide pheromone receptor. In yeast MATa cells lacking the endogenous Ste2p receptor, the S. macrospora PRE2 receptor facilitated all aspects of the pheromone response. Using a synthetic peptide, we can now predict the sequence of one active form of the S. macrospora peptide pheromone. We proved that S. macrospora wild-type strains secrete an active pheromone into the culture medium and that disruption of the ppg1 gene in S. macrospora prevents pheromone production. However, loss of the ppg1 gene does not affect vegetative growth or fertility. Finally, we established the yeast assay as an easy and useful system for analyzing pheromone production in developmental mutants of S. macrospora.     

The Pheromone Receptors Inhibit the Pheromone Response Pathway in Saccharomyces Cerevisiae by a Process That Is Independent of Their Associated Gα Protein

Dominant mutations at the DAF2 locus confer resistance to the cell-cycle arrest that normally occurs in MATa cells exposed to α-factor. One of these alleles, DAF2-2, has also been shown to suppress the constitutive signaling phenotype of null alleles of the gene encoding the α subunit of the G protein involved in pheromone signaling. These observations indicate that DAF2-2 inhibits transmission of the pheromone response signal. The DAF2-2 mutation has two effects on the expression of a pheromone inducible gene, FUS1. In DAF2-2 cells, FUS1 RNA is present at an increased basal level but is no longer fully inducible by pheromone. Cloning of DAF2-2 revealed that it is an allele of STE3, the gene encoding the a-factor receptor. STE3 is normally an α-specific gene, but is inappropriately expressed in a cells carrying a STE3(DAF2-2) allele. The two effects of STE3(DAF2-2) alleles on the pheromone response pathway are the result of different functions of the receptor. The increased basal level of FUS1 RNA is probably due to stimulation of the pathway by an autocrine mechanism, because it required at least one of the genes encoding a-factor. Suppression of a null allele of the G(α) subunit gene, the phenotype associated with the inhibitory function of STE3, was independent of a-factor. This suppression was also observed when the wild-type STE3 gene was expressed in a cells under the control of an inducible promoter. Inappropriate expression of STE2 in α cells was able to suppress a point mutation, but not a null allele, of the G(α) subunit gene. The ability of the pheromone receptors to block the pheromone response signal in the absence of the G(α) subunit indicates that these receptors interact with another component of the signal transduction pathway.     

Mutations in a gene encoding the alpha subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling.

Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected. One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identical to the gene GPA1 (also known as SCG1), recently shown to be highly homologous to genes encoding the alpha subunits of mammalian G proteins. Physiological analysis of temperature-sensitive gpa1 mutations suggests that the encoded G protein is involved in signaling in response to mating pheromones. Mutational disruption of G-protein activity causes cell-cycle arrest in G1, deposition of mating-specific cell surface agglutinins, and induction of pheromone-specific mRNAs, all of which are responses to pheromone in wild-type cells. In addition, mutants can conjugate without the benefit of mating pheromone or pheromone receptor. A model is presented where the activated G protein has a negative impact on a constitutive signal which normally keeps the pheromone response repressed.     

Genetic fine-structural analysis of the Saccharomyces cerevisiae alpha-pheromone receptor.

The alpha-pheromone receptor encoded by the STE2 gene contains seven potential transmembrane domains. Its ability to transduce the pheromone signal is thought to require the action of a G protein. As an initial step toward defining the structural features of the receptor required for its activity, we examined the phenotypic consequences of linker insertion mutations (12 bp) at 10 different sites in the STE2 gene. Three mutant classes, which correspond to three different regions of the receptor protein, were observed. 1) The two mutants affecting the C-terminal region (C-terminal mutants) were essentially wild type for mating efficiency, pheromone binding, and pheromone sensitivity. 2) The three mutants in the N-terminus mated with reduced efficiency, showed reduced pheromone binding capacity, and were partially defective in pheromone induction of agglutinin production and cell division arrest. Increased gene dosage of these N-terminal alleles suppressed their mutant phenotypes, whereas the sst2-1 mutation, which blocks adaptation to pheromone, did not result in suppression. Thus, the N-terminal mutants were apparently limited by receptor production, but not by the adaptation function SST2. 3) The five mutants in the central region containing the seven transmembrane segments (central mutants) were completely defective for mating and did not respond to pheromone, but could be distinguished by their ability to bind pheromone. Inserts in or near transmembrane domains 2 and 4 blocked pheromone binding, whereas inserts into transmembrane domains 1, 5, and 6 retained partial pheromone binding activity even though they failed to transduce a signal. The central mutants were not suppressed by increased gene dosage, and one mutant (ste2-/101) was partially suppressed by sst2-1. Furthermore, the central core mutants were also distinguished from one another in that three of the five mutants were able to partially complement the temperature sensitivity of ste2-3.     

杨柳田头菇B交配型基因信息素受体片段的克隆与分析

利用简并PCR及DNA步移法,从杨柳田头菇Agrocybe salicacola YAASM0711菌株中扩增得到了一个4 231 bp的核酸片段.经过比对及序列预测,所获得序列中含有杨柳田头菇交配型编码基因中的信息素受体部分,其序列长度为1194 bp,包含4个内含子,5个外显子的长度分别为217 bp,113 bp,67 bp,138bp,449 bp.拼接后的ORF全长984 bp,编码327个氨基酸残基.该序列与灰盖鬼伞Coprinus cinerea、双色蜡蘑Laccaria bicolor信息素受体氨基酸序列较为相似,含有7个跨膜区.信息素受体遗传进化分析显示,其与多个物种信息素受体聚集在一起,可能与真菌信息素受体的多种起源有关.     

Olfactory receptor responses of the nymphal American cockroach to sex pheromones and their mimics

1. EAG responses to highly purified sex pheromones (periplanone-A and -B), sex pheromone mimics [germacrene-D, (+)-verbanyl acetate and (+)-trans-verbenyl acetate] and general odor (camphor) were recorded from both sexes of adult and three nymphal stages (7, 10 and 11th instars) of the American cockroach. 2. The M/F ratios were evaluated for each stage by stimulation with the above chemicals. 3. The ratio values indicated undeveloped sex pheromone receptor on the antennae of 7th male instar and females of all the stages. On the other hand, precursory development of the receptor was expected on the antennae of males of the old-aged nymphal instars and full development on the adult male antennae.