担子菌交配型基因的克隆及功能研究进展

担子菌中已有２０多个交配型基因被克隆,Ａ、Ｂ位点的交配型基因具有明显不同的结构特征。Ａ位点的交配型基因编码两类含相似同源结构域的蛋白质,两种蛋白质形成异源二聚体,调节依赖Ａ因子的发育过程;而Ｂ位点的交配型基因编码信息素及其受体,交配型基因编码的同源结构域蛋白质在植物、动物和菌类中都具有广泛的保守性。     

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。     

甘蓝中BcpLH同源基因的克隆及其表达分析

根据大白菜ＢｃｐＬＨ基因设计引物,用ＰＣＲ方法从甘蓝结球期的茎尖组织中克隆到了大白菜ＢｃｐＬＨ基因的同源基因ＢｏＬＨ１。序列分析表明,ＢｏＬＨ１基因含有３个内含子,ｃＤＮＡ编码２４５个氨基酸,编码的蛋白中存在两个双链ＲＮＡ结合蛋白结构域;Ｓｏｕｔｈｅｒｎ杂交结果显示在甘蓝基因组有１个以上的ＢｏＬＨ１基因拷贝。ＰＣＲ表达分析表明,甘蓝ＢｏＬＨ１同源基因主要在结球期的茏尖组织、球叶和包叶中表达。推测Ｂ     

LIM同源框基因家族与神经系统

同源框基因是指一类含有同源序列的基因,它编码的蛋白质作为转录调节因子调节细胞的发育和分化,控制基因的表达形式。ＬＩＭ同源框基因不仅含有同源框基因也含有编码ＬＩＭ结构域的保守序列。     

毕赤酵母碳源阻遏因子编码基因PpMIG1和PpMIG2的克隆与序列分析

通过MIG基因的同源性设计简并引物,采用PCR方法从毕赤酵母(Pichia pastoris)中成功克隆了两个MIG同源基因的核心片段,同时利用Genome Walking的方法获得了基因的全长及其侧翼序列,并分别命名为PpMIG1和PpMIG2.序列分析显示,PpMIG1基因全长1 335 bp,编码444个氨基酸;PpMIG2基因全长1 365 bp,编码454个氨基酸.这个两个基因所编码的蛋白质均与酿酒酵母碳源阻遏因子ScMig1同源,在氨基末端具有两个典型的C2H2锌指结构域,该结构域能够与受葡萄糖阻遏的许多基因的启动子相结合.PpMig阻遏因子的获得为毕赤酵母碳源阻遏机制的研究奠定了基础.     

Wilson病基因产物及铜转运P型ATP酶的研究进展

肝豆状核变性（ＷＤ）基因已被克隆,序列分析表明,其基因产物（ＡＴＰ７Ｂ）编码的是一种衾中１４１１个氨基酸的铜转运Ｐ型ＡＴＰ酶。ＡＴＰ７Ｂ具有这些酶所独有的功能结构区：金属离子结合位点、阳离子转导区、ＳＥＨＰ序更及跨膜区等,ＷＤ基因高频突变位点多与这些结构有密切关系。对ＡＴＰ７Ｂ的深入研究将为探讨ＷＤ的发病机制及诊断和治疗提供帮助。     

毛木耳A交配型位点的克隆和序列结构分析

以毛木耳Auricularia cornea杂交子APM2-16的亲本单核体APP7和M2S16为研究材料,经同源序列比对,从单核体APP7的基因组中获得了A交配型位点序列,并定位了mip基因的位置。根据APM2-16的转录组和单核体APP7基因组的相关信息设计引物,通过PCR扩增和克隆测序的方式获得单核体APP7和M2S16中的HD基因序列,通过生物信息学分析发现两者在核苷酸序列上没有同源性,都编码同源结构域转录因子。通过对毛木耳交配型位点的研究有助于进一步开展该物种的遗传学基础研究,为菌种遗传改良奠定基础。     

单增李斯特菌hfq基因的克隆及分子特征分析

目的:了解单增李斯特菌(Listeria monocytogenes,LM)SB5野毒株ncRNA伴侣分子hfq基因及其编码蛋白质的分子生物学特征。方法:利用PCR方法对hfq基因进行扩增、克隆及测序,对hfq基因分子特征进行分析,预测其编码蛋白质的二三级结构及功能活性位点,对其进行同源性及遗传变异分析。结果:LM hfq基因全长234 bp,编码77个氨基酸,对推导的Hfq氨基酸序列分析发现从N端到C端包含1个α-螺旋及5个β-折叠,具有RNA结合位点及六聚体结合位点。LM-SB5 hfq基因核苷酸序列与李斯特菌属各菌株同源率为94.5~100%,与其他种属细菌同源率为36.19~62.39%。结论:Hfq蛋白具有RNA的结合位点,可能在细菌ncRNA调节基因表达过程中发挥重要作用。     

子囊菌交配型位点与交配型基因研究进展

有性生殖是真菌的生殖方式之一,是真菌遗传重组的重要驱动力。交配型(mating-type,MAT)位点控制真菌性别,在有性生殖过程中起决定性作用。不同类型真菌MAT位点的基因组成、排列方式和编码蛋白不尽相同。近年来,MAT位点和MAT基因的功能与调控网络研究进展较快。本文对子囊菌交配型位点的基因组成及分布、MAT基因的功能、MAT位点与有性生殖调控通路的关系等进行了综述。     

喜盐鸢尾Na~+/H~+逆转运蛋白基因IhNHX1的克隆及序列分析

采用同源克隆和RACE法克隆获得喜盐鸢尾(Iris halophila Pall.)Na+/H+逆转运蛋白基因IhNHX1的全长序列,该基因序列的全长为1 946 bp,包含1个长度为1 611 bp的开放阅读框(ORF),编码537个氨基酸。序列对比及系统树分析结果表明:IhNHX1基因编码的氨基酸序列与另外11种植物NHX1基因编码的氨基酸序列的一致性高达96.2%,相同序列占61.7%,表明该氨基酸序列保守性较高;在系统树上喜盐鸢尾与其他植物的分支长均大于1.2,表明它们的亲缘关系均较远;IhNHX1基因编码的氨基酸序列含有2个保守结构域,即氨氯吡嗪结合位点和CaM结合结构域,分别是NHX1蛋白的标志性结合位点和重要调节区域。该蛋白质的二级结构和跨膜结构域分析结果表明:在IhNHX1基因编码的蛋白质的二级结构中,α螺旋占48.60%、不规则卷曲占32.03%、延伸链占14.71%、氢键转角占4.66%;该蛋白质含有10个跨膜结构域。此外,对5’RACE方法中5’端正向引物的优化设计步骤进行了归纳,以提高PCR扩增的特异性。     

基于基因组数据解析大球盖菇交配型系统

大球盖菇Stropharia rugosoannulata是一种具有较强木质纤维素降解能力以及高营养的重要食用菌。此菌属于四极性异宗结合担子菌,但是交配型位点结构仍未被解析。本研究利用基因组数据通过生物信息学方法进行大球盖菇的A和B交配型位点解析,并与其他大型真菌进行比较。结果显示大球盖菇的A交配型位点包含了一对保守的HD1和HD2基因,位点上下游基因的共线性较高,与Galerina patagonica和砖红韧黑伞Hypholoma sublateritium相似度最高,A位点结构上也较为保守,在上下游具有保守的MIP、Sec61蛋白、甘氨酸脱氢酶和β侧翼蛋白。B位点包含5个信息素受体和3个信息素前体基因,与其他真菌相比较发现,B位点及上下游基因的共线性较差,表明在不同的真菌中B位点变异性较大。本研究所获得的结果将有助于诠释大球盖菇交配型位点结构,为今后的遗传育种提供了理论依据。     

Identification of mating type loci and development of SCAR marker genetically linked to the B3 locus in Pleurotus eryngii

In order to estimate how diverse the mating types in Pleurotus eryngii from different regions are, pairings between monokaryons derived from inter- and intragroups were done. Sixteen and 15 alleles were identified at loci A and B from the 12 strains. In the P. eryngii KNR2312, widely used for commercial production, four mating loci, A3, A4, B3, and B4, were determined. Those loci, except A3, were found in 4 strains out of 12 strains. To improve breeding efficiency, especially in mating type determination, RAPD and BSA were performed to screen for a mating type specific marker. The SCAR marker 13- 2(2100) was developed based on the RAPD-derived sequence typing B3 locus. The sequence analysis of 13-2(2100) revealed that it contained a conserved domain, the STE3 superfamily, and consensus sequences like the TATA box and GC box. It seems likely that the SCAR marker region is a part of the pheromone receptor gene.     

The pheromone response factor coordinates filamentous growth and pathogenicity in Ustilago maydis.

In Ustilago maydis, the a and b mating type loci regulate cell fusion, filamentous growth and pathogenicity. The a locus encodes a pheromone-based cell recognition system, and the b locus specifies two homeodomain proteins. The expression of all genes in the a and b loci is induced by pheromone. We have identified a HMG protein (Prf1) that binds sequence specifically to pheromone response elements present in the a and b loci. prf1 mutants do not express the a and b genes and are sterile. The disruption of prf1 in pathogenic haploid strains results in a loss of pathogenicity. The constitutive expression of the b genes restores pathogenicity and induces filamentous growth in the absence of the pheromone signal. These results provide evidence that pheromone signalling, filamentous growth and pathogenic development are linked through Prf1.     

The Neurospora crassa pheromone precursor genes are regulated by the mating type locus and the circadian clock

Pheromones play important roles in female and male behaviour in the filamentous ascomycete fungi. To begin to explore the role of pheromones in mating, we have identified the genes encoding the sex pheromones of the heterothallic species Neurospora crassa. One gene, expressed exclusively in mat A strains, encodes a polypeptide containing multiple repeats of a putative pheromone sequence bordered by Kex2 processing sites. Strains of the opposite mating type, mat a, express a pheromone precursor gene whose polypeptide contains a C-terminal CAAX motif predicted to produce a mature pheromone with a C-terminal carboxy-methyl isoprenylated cysteine. The predicted sequences of the pheromones are remarkably similar to those encoded by other filamentous ascomycetes. The expression of the pheromone precursor genes is mating type specific and is under the control of the mating type locus. Furthermore, the genes are highly expressed in conidia and under conditions that favour sexual development. Both pheromone precursor genes are also regulated by the endogenous circadian clock in a time-of-day-specific fashion, supporting a role for the clock in mating.     

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。     

A large pheromone and receptor gene complex determines multiple B mating type specificities in Coprinus cinereus.

Pheromone signaling plays an essential role in the mating and sexual development of mushroom fungi. Multiallelic genes encoding the peptide pheromones and their cognate 7-transmembrane helix (7-TM) receptors are sequestered in the B mating type locus. Here we describe the isolation of the B6 mating type locus of Coprinus cinereus. DNA sequencing and transformation analysis identified nine genes encoding three 7-TM receptors and six peptide pheromone precursors embedded within 17 kb of mating type-specific sequence. The arrangement of the nine genes suggests that there may be three functionally independent subfamilies of genes each comprising two pheromone genes and one receptor gene. None of the nine B6 genes showed detectable homology to corresponding B gene sequences in the genomic DNA from a B3 strain, and each of the B6 genes independently alter B mating specificity when introduced into a B3 host strain. However, only genes in two of the B6 groups were able to activate B-regulated development in a B42 host. Southern blot analysis showed that these genes failed to cross-hybridize to corresponding genes in the B42 host, whereas the three genes of the third subfamily, which could not activate development in the B42 host, did cross-hybridize. We conclude that cross-hybridization identifies the same alleles of a particular subfamily of genes in different B loci and that B6 and B42 share alleles of one subfamily. There are an estimated 79 B mating specificities: we suggest that it is the different allele combinations of gene subfamilies that generate these large numbers.