耐低温酵母Curvibasidium rogersii菌株在松萝样品中的首次分离鉴定及基于基因组分析的生物特性探究

【目的】对西藏松萝地衣来源的两株非常规酵母进行分离鉴定,并通过基因组序列分析探究其生物学特性和应用潜力。【方法】从西藏来源的松萝地衣样品内部分离得到2株耐低温酵母菌株,通过26S D1/D2和ITS序列比对分析以及生理生化实验进行菌种鉴定;通过全基因组序列分析和验证探究菌株的生物特性。【结果】两株酵母菌株经鉴定均为Curvibasidium rogersii,可以在10℃低温良好生长,在20℃生长最佳,25℃及以上温度生长缓慢或不生长。对其进行基因组测序和基因组挖掘,测序结果发现,其基因组注释出功能的部分与产油脂的低温酵母白冬孢酵母Leucosporidium creatinivorum具有最高相似性,尼罗红染色发现两株酵母都能够生产油脂,另外在基因组序列中还发现了可能参与木糖代谢的相关蛋白编码基因,实验证明两个酵母菌株可以利用木糖生长。【结论】首次分离鉴定了来自西藏松萝的酵母C. rogersii,为充分开发利用松萝和其他地衣来源微生物,以及利用可代谢木糖的新资源酵母生产微生物油脂提供了基础。     

水稻基腐病细菌毒素的遗传特性和产毒相关的分子标记

[目的]水稻基腐病(Erwinia chrysanthemi pv.zeae)是水稻上重要的细菌病害之一,本论文对该病菌的毒素遗传特性和产毒相关的分子标记进行了研究.[方法]通过化学诱变方法,筛选基腐细菌去质粒的突变体Ech7-mu1;应用RAPD技术,筛选产毒素相关的分子标记.[结果]毒素活性测定结果表明,野生菌Ech7和去质粒菌株Ech7-mu1都能产生毒素.从260条随机引物中,筛选出引物K10,该引物能从不产生毒素的突变株Ech7-4中扩增出大小为2139bp的DNA特异片段,但不能扩增野生菌Ech7,将该片段克隆,测序分析,设计特异引物,在突变体Ech7-4中获得了与毒素产生相关的SCAR分子标记(标记符合率为100%).该基因片段有5个ORFs,其中2个ORFs分别编码NADH-黄素还原酶和N-乙酰转移酶,另外2个不完整的ORFs编码的蛋白分别与Pseudomonas aerginosa(ZP00136947)和Yersinia Pestis(ZP01177873)的抗菌素代谢转运蛋白通透酶(DMT)具有66%和46%的同源率.[结论]水稻基腐细菌毒素的生物合成是由染色体基因编码,与质粒无关.不产生毒素的突变菌株基因突变的位点位于SCAR标记DNA的3'末端.     

Molecular Characterization of Phytoplasma Associated with Rose Witches'-Broom in China

In 2005, rose plants (Rosa rugosa cv. ‘Plena’) exhibiting typical phytoplasma disease symptoms of stunting, yellowing, witches’‐broom and dieback were observed in Pingyin, Shandong Province, China. The disease, rose witches’‐broom (RoWB), is progressively destructive and can be graft‐transmitted. Polymerase chain reaction (PCR), sequencing of PCR products and electron microscopy were used to investigate the possible association of phytoplasma with RoWB. All results indicated that presence of phytoplasma in the symptomatic rose plants. Sequence alignment of 16S rRNA gene, tuf gene and rp gene confirmed that the phytoplasma associated with RoWB is the causal agent of Paulownia witches’‐broom disease, which might be transmitted from the paulownia tree that is several meters away. To our knowledge, this is the first report of the molecular characterization of phytoplasma infecting rose in China.     

Regulation of Transient Site-specific Copy Gain by MicroRNA

Intra-tumor copy number heterogeneity is commonly observed in cancer; however, the molecular mechanisms that contribute to heterogeneity remain poorly understood. Up-regulation of the histone demethylase KDM4A promotes transient site-specific copy gain (TSSG) in cells; therefore, uncovering how KDM4A levels are controlled is important for understanding the regulation of copy number heterogeneity. Here, we demonstrate that KDM4A is regulated by hsa-mir-23a-3p, hsa-mir-23b-3p, and hsa-mir-137. Altering expression of these microRNAs (miRNAs) regulates KDM4A-dependent TSSG. miRNA inhibition promoted copy gains and increased expression of the drug-resistant oncogene CKS1B, which was further substantiated in primary breast tumors. Consistent with increased CKS1B expression, miRNA inhibition reduced breast cancer cell sensitivity to cisplatin. Our data identify these miRNAs as regulators of TSSG and copy gains of a drug resistance gene.     

Identification of the receptor binding domain of the mouse mammary tumor virus envelope protein

Mouse mammary tumor virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse transferrin receptor 1 for cell entry. To characterize the interaction of MMTV with its receptor, we aligned the MMTV envelope surface (SU) protein with that of Friend murine leukemia virus (F-MLV) and identified a putative receptor-binding domain (RBD) that included a receptor binding sequence (RBS) of five amino acids and a heparin-binding domain (HBD). Mutation of the HBD reduced virus infectivity, and soluble heparan sulfate blocked infection of cells by wild-type pseudovirus. Interestingly, some but not all MMTV-like elements found in primary and cultured human breast cancer cell lines, termed h-MTVs, had sequence alterations in the putative RBS. Single substitution of one of the amino acids found in an h-MTV RBS variant in the RBD of MMTV, Phe(40) to Ser, did not alter species tropism but abolished both virus binding to cells and infectivity. Neutralizing anti-SU monoclonal antibodies also recognized a glutathione S-transferase fusion protein that contained the five-amino-acid RBS region from MMTV. The critical Phe(40) residue is located on a surface of the MMTV RBD model that is distant from and may be structurally more rigid than the region of F-MLV RBD that contains its critical binding site residues. This suggests that, in contrast to other murine retroviruses, binding to its receptor may result in few or no changes in MMTV envelope protein conformation.