拟南芥和水稻金属蛋白酶ftsH基因家族的基因组比较分析

FtsH(Filamentation temperature-sensitive H)是一种广泛存在于原核生物和真核生物中的ATP依赖型金属蛋白酶。同源性分析表明,在拟南芥和水稻基因组中分别有12个和9个ftsH基因。ftsH基因在染色体上的分布有明显的偏爱性,如拟南芥的1、2、5号染色体和水稻的1、5号染色体。亚细胞定位分析表明,所有FtsH蛋白均定位于叶绿体或线粒体中。系统进化分析表明,21个FtsH蛋白成员可分为8个类群,其中AtFtsH12在水稻中没有发现种间同源物。每个类群成员的蛋白序列高度保守,种内同源物显示出大于80%的相似性,而种间同源物的相似性也大于70%。类群内的同源基因并非平行进化产生的,拟南芥基因组中进化出AtftsH1/5、AtftsH2/8、AtftsH3/10和AtftsH7/9共4个同源基因对,而水稻基因组中只有OsftsH3/8和OsftsH4/5两个同源基因对。每一类群中的成员在基因外显子-内含子边界分布上表现出高度保守性,在蛋白功能结构域的可变残基上具有偏爱性,而内含子在碱基组成和序列长度上表现出广泛的变异。拟南芥和水稻ftsH基因家族的比较分析为其他物种ftsH基因的特...     

RNA isolation from yeast using silica matrices.

RNA isolation from yeast is complicated by the need to initially break the cell wall. While this can be accomplished by glass bead disruption or enzyme treatment, these approaches result in DNA contamination and/or the need for incubation periods. We have developed a protocol for the isolation of RNA samples from yeast that minimizes degradation by RNases and incorporates two purification steps: acid phenol extraction and binding to a silica matrix. The procedure requires no precipitation steps, facilitating automation, and can be completed in less than 90 min. The RNA quality is ideal for microarray analysis.     

Saturation mutagenesis of a +1 programmed frameshift-inducing mRNA sequence derived from a yeast retrotransposon

Errors during the process of translating mRNA information into protein products occur infrequently. Frameshift errors occur less frequently than other types of errors, suggesting that the translational machinery has more robust mechanisms for precluding that kind of error. Despite these mechanisms, mRNA sequences have evolved that increase the frequency up to 10,000-fold. These sequences, termed programmed frameshift sites, usually consist of a heptameric nucleotide sequence, at which the change in frames occurs along with additional sequences that stimulate the efficiency of frameshifting. One such stimulatory site derived from the Ty3 retrotransposon of the yeast Saccharomyces cerevisiae (the Ty3 stimulator) comprises a 14 nucleotide sequence with partial complementarity to a Helix 18 of the 18S rRNA, a component of the ribosome's accuracy center. A model for the function of the Ty3 stimulator predicts that it base pairs with Helix 18, reducing the efficiency with which the ribosome rejects erroneous out of frame decoding. We have tested this model by making a saturating set of single-base mutations of the Ty3 stimulator. The phenotypes of these mutations are inconsistent with the Helix 18 base-pairing model. We discuss the phenotypes of these mutations in light of structural data on the path of the mRNA on the ribosome, suggesting that the true target of the Ty3 stimulator may be rRNA and ribosomal protein elements of the ribosomal entry tunnel, as well as unknown constituents of the solvent face of the 40S subunit.     

Oxygen stress: a regulator of apoptosis in yeast.

Oxygen radicals are important components of metazoan apoptosis. We have found that apoptosis can be induced in the yeast Saccharomyces cerevisiae by depletion of glutathione or by low external doses of H2O2. Cycloheximide prevents apoptotic death revealing active participation of the cell. Yeast can also be triggered into apoptosis by a mutation in CDC48 or by expression of mammalian bax. In both cases, we show oxygen radicals to accumulate in the cell, whereas radical depletion or hypoxia prevents apoptosis. These results suggest that the generation of oxygen radicals is a key event in the ancestral apoptotic pathway and offer an explanation for the mechanism of bax-induced apoptosis in the absence of any established apoptotic gene in yeast.     

基于全长转录组的蛇足石杉ARF基因家族鉴定分析

生长素响应因子(ARF)是介导生长素信号传导并调节多种生物学过程的转录因子家族。为探究蛇足石杉ARF基因家族成员及其在高温和干旱胁迫响应中的作用,该研究利用全长转录组和RNA-seq数据,分析蛇足石杉ARF基因家族成员的系统发育及表达模式,并通过生物信息学分析,对ARF基因家族的理化性质、结构域、保守基序、系统发育、组织表达模式及高温和干旱胁迫下的表达模式进行分析。结果表明:(1)在蛇足石杉全长转录组中共筛选出24个ARF家族成员,均为酸性蛋白且均为亲水蛋白。(2)亚细胞定位预测显示,所有24个HsARF均定位于细胞核。(3)系统发育分析发现,HsARF与被子植物拟南芥和水稻亲缘关系较远,与高等开花植物只有2个共同的ARF祖先。(4)结构域分析发现,除HsARF18/23/24外,大多数HsARF有B3结构域。二级结构分析发现,HsARF蛋白占比最多的为无规卷曲,其次为延伸链和α-螺旋。24个HsARF蛋白中所用的三级结构模型只有4种。(5)RNA-seq分析显示,7个HsARF在所有检测的组织中表达量均较高,10个HsARF在茎中的表达量高于根和叶,而HsARF13和HsARF14在叶中的表达量低于根和茎。(6)HsARF在高温和干旱胁迫下表达量发生显著变化,其中18个HsARF基因不同程度高温胁迫诱导,有7个HsARF基因响应干旱胁迫,其中有3个HsARF基因受干旱诱导,有4个HsARF基因受干旱抑制。该研究结果为蛇足石杉HsARF基因的功能和生物育种提供了理论参考。     

水稻突触融合相关蛋白基因(OsKNOLLE)在酵母中的异源表达及在非生物胁迫中的作用初探

突触融合相关蛋白属于多功能蛋白家族SNARE(SolubleN-ethylmaleimide-sensitive factor attachment pro-tein receptor)的亚家族蛋白,这类家族在植物的许多生理过程中都起着非常重要的作用。为探明水稻突触融合相关蛋白OsKNOLLE(Syntaxin-related protein KNOLLE)的功能,文章从"中花11"水稻根中克隆OsKNOLLE基因的CDS序列并构建到酵母表达载体pYX212上,同时转化酿酒酵母菌株BY4741。通过比较不同酵母转化子在多种非生物因子胁迫下的表型,显示转OsKNOLLE酵母菌比对照菌适应逆境能力更强,表明OsKNOLLE与盐、Cu2+、H2O2、Cd2+、Hg2+胁迫相关,推测OsKNOLLE在植物对逆境的耐性中发挥重要作用。本研究为OsKNOLLE基因的研究提供方法支持,同时明确了其与不同非生物因子的相关性。     

Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana

High-salinity, drought, and low temperature are three common environmental stress factors that seriously influence plant growth and development worldwide. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators that have also been linked to stress responses. However, the relationship between miRNA expression and stress responses is just beginning to be explored. Here, we identified 14 stress-inducible miRNAs using microarray data in which the effects of three abiotic stresses were surveyed in Arabidopsis thaliana. Among them, 10 high-salinity-, four drought-, and 10 cold-regulated miRNAs were detected, respectively. miR168, miR171, and miR396 responded to all of the stresses. Expression profiling by RT-PCR analysis showed great cross-talk among the high-salinity, drought, and cold stress signaling pathways. The existence of stress-related elements in miRNA promoter regions provided further evidence supporting our results. These findings extend the current view about miRNA as ubiquitous regulators under stress conditions.     

Catharanthus roseus mitogen-activated protein kinase 3 confers UV and heat tolerance to Saccharomyces cerevisiae

Catharanthus roseus is an important source of pharmaceutically important Monoterpenoid Indole Alkaloids (MIAs). Accumulation of many of the MIAs is induced in response to abiotic stresses such as wound, ultra violet (UV) irradiations, etc. Recently, we have demonstrated a possible role of CrMPK3, a C. roseus mitogen-activated protein kinase in stress-induced accumulation of a few MIAs. Here, we extend our findings using Saccharomyces cerevisiae to investigate the role of CrMPK3 in giving tolerance to abiotic stresses. Yeast cells transformed with CrMPK3 was found to show enhanced tolerance to UV and heat stress. Comparison of CrMPK3 and SLT2, a MAPK from yeast shows high-sequence identity particularly at conserved domains. Additionally, heat stress is also shown to activate a 43 kDa MAP kinase, possibly CrMPK3 in C. roseus leaves. These findings indicate the role of CrMPK3 in stress-induced MIA accumulation as well as in stress tolerance.