Identification of five small heat shock protein genes in Spodoptera frugiperda and expression analysis in response to different environmental stressors

Spodoptera frugiperda (J. E. Smith) is a highly adaptable polyphagous migratory pest in tropical and subtropical regions. Small heat shock proteins (sHsps) are molecular chaperones that play important roles in the adaptation to various environment stressors. The present study aimed to clarify the response mechanisms of S. frugiperda to various environmental stressors. We obtained five S. furcifera sHsp genes (SfsHsp21.3, SfsHsp20, SfsHsp20.1, SfsHsp19.3, and SfsHsp29) via cloning. The putative proteins encoded by these genes contained a typical α-crystallin domain. The expression patterns of these genes during different developmental stages, in various tissues of male and female adults, as well as in response to extreme temperatures and UV-A stress were studied via real-time quantitative polymerase chain reaction. The results showed that the expression levels of all five SfsHsp genes differed among the developmental stages as well as among the different tissues of male and female adults. The expression levels of most SfsHsp genes under extreme temperatures and UV-A-induced stress were significantly upregulated in both male and female adults. In contrast, those of SfsHsp20.1 and SfsHsp19.3 were significantly downregulated under cold stress in male adults. Therefore, the different SfsHsp genes of S. frugiperda play unique regulatory roles during development as well as in response to various environmental stressors.     

茶树DELLA基因家族的鉴定及表达分析

利用生物信息学方法,从茶树（Camellia sinensis（L.）O.Ktze.）全基因组数据库中分析获得DELLA蛋白的家族成员,并对它们的系统进化关系、蛋白序列特征、基因表达特异性及其与茶树次生代谢物的相关性进行分析。结果显示：茶树基因组中共有5个DELLA基因,分别为：TEA009882（CsGAI）、TEA022818（CsRGA1）、TEA010112（CsRGL1）、TEA008736（CsRGL2）和TEA020933（CsRGL3）;其编码的氨基酸数量在525~594之间,均定位于细胞核。该蛋白的二级和三级结构分析结果表明,茶树DELLA蛋白结构中含有大量的α螺旋及少量β转角结构。蛋白保守结构域分析结果显示该蛋白与拟南芥（Arabidopsis thaliana（L.）Heynh.）具有高度的同源性,均具有GRAS、DELLA等保守结构域。基因的表达特异性分析结果表明,在茶树不同组织部位中,TEA009882、TEA022818和TEA010112基因的表达量均较高,而TEA020933和TEA008736的表达量在各组织中均较低;茶树DELLA基因的表达受到干旱、NaCl、低温及茉莉酸甲酯等非生物逆境胁迫的调控,且其表达量与茶树次生代谢物的积累间存在相关性。推测茶树DELLA基因广泛参与了茶树生长发育及非生物逆境胁迫的响应,以及对次生代谢物生物合成过程的调控。     

Pirh2

Ubiquitylation is currently recognized as a major posttranslational modification that regulates diverse cellular processes. Pirh2 is a ubiquitin E3 ligase that regulates the turnover and functionality of several proteins involved in cell proliferation and differentiation, cell cycle checkpoints, and cell death. Here we review the role of Pirh2 as a regulator of the DNA damage response through the ubiquitylation of p53, Chk2, p73, and PolH. By ubiquitylating these proteins, Pirh2 regulates cell cycle checkpoints and cell death in response to DNA double-strand breaks or the formation of bulky DNA lesions. We also discuss how Pirh2 affects cell proliferation and differentiation in unstressed conditions through ubiquitylation and degradation of c-Myc, p63, and p27^kip1. Finally, we link these different functions of Pirh2 to its role as a tumor suppressor in mice and as a prognosis marker in various human cancer subtypes.     

柠条锦鸡儿CkLEA1基因克隆及表达分析

植物受到逆境胁迫后,大量逆境响应基因会被诱导表达,LEA蛋白编码基因就是与植物抗旱、抗冷等非生物胁迫密切相关的一类基因.从已构建的柠条锦鸡儿干旱胁迫抑制性削减杂交文库中筛选到了一条LEA蛋白编码基因并进行了克隆.序列比对与系统进化分析显示该基因属于LEA3基因家族成员,命名为CkLEA1(GenBank登录号是KC309408).克隆得到该基因gDNA长469bp,包含两个外显子和一个内含子;cDNA长357bp,包含300bp的开放阅读框,推导编码99个氨基酸的蛋白质.利用荧光定量PCR技术对CkLEA1基因在各种逆境胁迫条件的表达情况进行初步研究表明,CkLEA1受干旱、ABA、冷、热、盐和碱等处理不同程度地诱导,推测其与柠条锦鸡儿响应逆境胁迫的机制有关.     

重组人源性脂联素球状结构的表达、纯化与活性检测

目的:利用基因工程方法构建人源性脂联素球状结构(gAd)基因的高效原核表达体系,并对重组蛋白进行诱导表达、纯化、鉴定及活性检测.方法:从正常人脂肪组织里面提取总RNA,反转录合成cDNA,经PCR扩增、酶切后连入pET-22b(+)载体构建重组质粒pET-22b(+)-gAd,重组质粒转化大肠杆菌BL21(DE3)感受态细胞.经诱导剂诱导后目的蛋白以包涵体形式产生,采用强碱促溶包涵体并用丙酮沉淀蛋白的方法进行复性和纯化,得到高纯度的人源性gAd.运用SDS-PAGE、Western blotting对重组蛋白进行鉴定,通过对蛋白激酶(AMPK)的磷酸化水平和对小鼠的心肌缺血再灌注损伤的保护作用来检测纯化蛋白的生物学活性.结果:成功构建了原核表达载体pET-22b(+)-gAd,实现了人源性gAd在原核细胞中的表达,并对形成的包涵体变性、复性和纯化,纯化出的蛋白经过SDS-PAGE和Western分析证实为gAd;通过对AMPK的磷酸化水平的检测和对小鼠的心肌缺血再灌注损伤的保护作用证明纯化出的gAd具有高生物学活性.结论:成功构建、表达和纯化了无标签、高生物学活性的人源性脂联素球状结构(gAd),为其进一步的理论研究、生产开发奠定了基础.     

Overcoming resistance to rapalogs in gliomas by combinatory therapies

Glioblastoma is the most common and aggressive brain tumor type, with a mean patient survival of approximately 1 year. Many previous analyses of the glioma kinome have identified key deregulated pathways that converge and activate mammalian target of rapamycin (mTOR). Following the identification and characterization of mTOR-promoting activity in gliomagenesis, data from preclinical studies suggested the targeting of mTOR by rapamycin or its analogs (rapalogs) as a promising therapeutic approach. However, clinical trials with rapalogs have shown very limited efficacy on glioma due to the development of resistance mechanisms. Analysis of rapalog-insensitive glioma cells has revealed increased activity of growth and survival pathways compensating for mTOR inhibition by rapalogs that are suitable for therapeutic intervention. In addition, recently developed mTOR inhibitors show high anti-glioma activity. In this review, we recapitulate the regulation of mTOR signaling and its involvement in gliomagenesis, discuss mechanisms resulting in resistance to rapalogs, and speculate on strategies to overcome resistance. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).