植物非生物逆境胁迫相关RING finger蛋白

RING finger蛋白是锌指蛋白类中一个庞大的蛋白家族,已成为拟南芥第三大蛋白家族,在水稻中也发现了488个。最典型的结构特点是序列内包含环指结构域(RING finger domain)。RING finger蛋白主要通过泛素化途径参与到植物细胞的生理生化过程。介绍了植物RING finger蛋白的结构特点和分类,综述它们的亚细胞定位,重点阐述它们在响应干旱胁迫、温度胁迫和盐胁迫等非生物逆境胁迫的调控作用。     

沙田柚RING-H2 finger基因的生物信息学分析

为探讨沙田柚自交不亲和性的机理,利用高通量测序技术对沙田柚自交和异交花柱进行转录组测序,通过差异分析得到了沙田柚RING-H2 finger基因序列。采用生物信息学方法,对其编码的蛋白质从序列特征、理化性质、跨膜结构域、高级结构以及功能域等方面进行了预测和分析。结果表明:该基因全长为845 bp,开放阅读框(ORF)全长为387 bp,编码128个氨基酸,编码蛋白质的分子量为13.61 KDa,理论等电点为4.26;该蛋白质含有一个植物RING-H2 finger蛋白家族的保守结构域,为疏水性不稳定蛋白。氨基酸序列分析表明,其编码的氨基酸与甜橙(Cs4g15340.1)RING-H2 finger蛋白的相似度为98%。这些分析结果可为今后深入研究该蛋白的结构特征和功能提供参考。     

Expression,purification, and characterization of N-terminal His-tagged proteins with mutations in zinc finger 3 of zinc finger protein ZNF191(243–368)

Abstract

Zinc finger protein ZNF191(243–368), the zinc finger region of ZNF191, is potentially associated with cell proliferation in hepatocellular carninoma. A His-tag expression system was used to express and purify proteins with mutations in the zinc finger 3 of ZNF191(243–368) for analysis of protein properties, structure, and functions. The purification of the His-tag fusion proteins was simpler and faster than that of the ZNF191(243–368) inclusion bodies. The properties and structures of the His-tag fusion mutant proteins were investigated using spectrographic techniques and DNA hydrolysis experiment. The His₆-tag system could be used to express ZNF191(243–368). The presence of the His₆-tag at the N-terminus of ZNF191(243–368) did not evidently affect its properties and structure. However, the site-directed mutations in zinc finger 3 affected the structure of the protein. The DNA hydrolase activity of His₆-ZF-F3/H4 suggested that four histidines in zinc finger 3 might form a structure similar to that of the active center in a hydrolase. This work reports that continuous histidines need to form a certain structure for specific functions, and provides new insights into the design of an artificial nuclease.     

胡杨Ring finger E3连接酶PeRH2提高烟草耐旱机制研究

近期研究表明泛素化在植物胁迫响应中扮演一个关键的角色。作为泛素-蛋白酶体系统(UPS)中的关键酶,泛素E3连接酶更是在细胞的新陈代谢中起着重要的作用。本文从胡杨中克隆了一个C端含有Ring finger结构域的蛋白(152个氨基酸)编码基因(Pe RH2),经过序列比对推断该基因编码产物为泛素E3连接酶,属于Ring finger家族中的Ring-H2构型。研究显示,甘露醇胡杨苗木在255 mmol/L甘露醇胁迫下,叶片Pe RH2表达量上调。拟南芥原生质体中瞬时表达融合质粒p Yellow0029-Pe RH2的研究表明,Pe RH2蛋白定位于细胞核。以农杆菌介导法将Pe RH2转入烟草,Sq RT-PCR结果显示,Pe RH2在转基因烟草中获得过表达。在255 mmol/L甘露醇处理下,转基因烟草幼苗耐受渗透胁迫的能力高于野生型和转空载体烟草。干旱处理后,转基因烟草的叶片保水力明显高于野生型和转空载体烟草,而叶片相对电导率、MDA的含量也要低于野生型和转空载体烟草。因此,过表达Pe RH2能够提高烟草的抗旱能力。     

Zinc finger motif for single-stranded nucleic acids? investigations by nuclear magnetic resonance

Nuclear magnetic resonance (NMR) methods have been used to address issues regarding the relevance and feasibility of zinc binding to "zinc finger-like" sequences of the type C-X2-C-X4-H-X4-C [referred to as CCHC or retroviral-type (RT) zinc finger sequences]. One-dimensional (1D) NMR experiments with an 18-residue synthetic peptide containing the amino acid sequence of an HIV-1 RT-zinc finger domain (HIV1-F1) indicate that the sequences are capable of binding zinc tightly and stoichiometrically. 1H-113Cd spin echo difference NMR data confirm that the Cys and His amino acids are coordinated to metal in the 113Cd adduct. The 3D structure of the zinc adduct [Zn(HIV1-F1)] was determined to high atomic resolution by a new NMR-based approach that utilizes 2D-NOESY back-calculations as a measure of the consistency between the structures and the experimental data. Several interesting structural features were observed, including (1) the presence of extensive internal hydrogen bonding, and (2) the similarity of the folding of the first six residues to the folding observed by X-ray crystallography for related residues in the iron domain of rubredoxin. Structural constraints associated with conservatively substituted glycines provide further rationale for the physiological relevance of the zinc adduct. Similar NMR and structural results have been obtained for the second HIV-1 RT-zinc finger peptide, Zn(HIV1-F2). NMR studies of the zinc adduct with the NCP isolated directly from HIV-1 particles provide solid evidence that zinc finger domains are formed that are conformationally similar (if not identical) to the peptide structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

PHD finger protein 8蛋白多克隆抗体的制备、纯化与检测

PHD finger8(PHF8)蛋白是最新发现的一种带有PHD结构域和Jmjc结构域的蛋白。现有研究表明其可能在基因转录、组蛋白去甲基化等方面发挥重要作用。为研究其功能,本研究构建原核表达载体pET41b-PHF8(aa886-936),在大肠杆菌Escherichia coli BL21中诱导表达带有GST标签的PHF8(aa886-936)亲水片段融合蛋白,并纯化该片段作为抗原免疫家兔,再以CNBr活化Sepharose4B微珠纯化抗血清制备PHF8特异性多克隆抗体。Western blotting以及免疫荧光检测表明该抗体具有很好的特异性,同时免疫荧光染色的结果也表明PHF8蛋白定位于细胞核。     

Site-specific cleavage of DNA–RNA hybrids by zinc finger/FokI cleavage domain fusions

Zinc-finger proteins of the Cys₂His₂ type bind DNA–RNA hybrids with affinities comparable to those for DNA duplexes. Such zinc-finger proteins were converted into site-specific cleaving enzymes by fusing them to the FokI cleavage domain. The fusion proteins are active and under optimal conditions cleave DNA duplexes in a sequence-specific manner. These fusions also exhibit site-specific cleavage of the DNA strand within DNA–RNA hybrids albeit at a lower efficiency (≃50-fold) compared to the cleavage of the DNA duplexes. These engineered endonucleases represent the first of their kind in terms of their DNA–RNA cleavage properties, and they may have important biological applications.     

In-silico mining,type and frequency analysis of genic microsatellites of finger millet (Eleusine coracana (L.) Gaertn.): a comparative genomic analysis of NBS–LRR regions of finger millet with rice

In recent years, the increased availability of the DNA sequences has given the possibility to develop and explore the expressed sequence tags (ESTs) derived SSR markers. In the present study, a total of 1956 ESTs of finger millet were used to find the microsatellite type, distribution, frequency and developed a total of 545 primer pairs from the ESTs of finger millet. Thirty-two EST sequences had more than two microsatellites and 1357 sequences did not have any SSR repeats. The most frequent type of repeats was trimeric motif, however the second place was occupied by dimeric motif followed by tetra-, hexa- and penta repeat motifs. The most common dimer repeat motif was GA and in case of trimeric SSRs, it was CGG. The EST sequences of NBS-LRR region of finger millet and rice showed higher synteny and were found on nearly same positions on the rice chromosome map. A total of eight, out of 15 EST based SSR primers were polymorphic among the selected resistant and susceptible finger millet genotypes. The primer FMBLEST5 could able to differentiate them into resistant and susceptible genotypes. The alleles specific to the resistant and susceptible genotypes were sequenced using the ABI 3130XL genetic analyzer and found similarity to NBS–LRR regions of rice and finger millet and contained the characteristic kinase-2 and kinase 3a motifs of plant R-genes belonged to NBS–LRR region. The In-silico and comparative analysis showed that the genes responsible for blast resistance can be identified, mapped and further introgressed through molecular breeding approaches for enhancing the blast resistance in finger millet.