硝磺草酮、铜单一和复合胁迫对苦草的生长及生理的影响

农药与重金属是水体中常见的污染物，其在水环境中的污染问题已引起全社会的广泛关注。目前关于农药硝磺草酮与铜的研究主要集中在动物与浮游植物上，对大型水生植物的研究较匮乏。为了阐明水体中常见毒性污染物硝磺草酮与铜对水生植物的潜在毒性作用，本研究探究了二者对长江流域的优势种苦草（Vallisneria natans）的生长及生理影响，旨在为水生植物复合污染的生态毒性效应和生态安全评估提供依据。本研究采用水培法，研究了不同浓度硝磺草酮（0、0.01、1、10、20、50 mg/L）、铜（0、0.1、0.3、0.5、1、2 mg/L）以及（硝磺草酮+铜）在（0+0、0.01+0.1、1+0.3、10+0.5、20+1、50 mg/L+2 mg/L）浓度时对苦草的相对生长率、光合色素（叶绿素a、叶绿素b、类胡萝卜素）、抗氧化酶〔超氧化物歧化酶（superoxide dismutase， SOD）、过氧化物酶（peroxidase， POD）、过氧化氢酶（catalase， CAT）〕以及可溶性蛋白含量变化的影响。结果显示，单一硝磺草酮对苦草的生长和光合色素的合成、CAT活性和可溶性蛋白含量具有抑制作用，而对苦草POD活性具有激活效应；单一铜胁迫对苦草生长、光合色素的合成、CAT活性以及可溶性蛋白含量具有显著抑制作用，而对苦草SOD和POD活性具有激活效应；此外，苦草的相对生长率、光合色素、可溶性蛋白含量、CAT活性等指示物对硝磺草酮与铜联合胁迫表现出受害响应，而POD活性显著上升，SOD活性呈现低浓度抑制高浓度促进效应。毒性效应评估结果显示，随着复合胁迫浓度的升高，硝磺草酮和铜对苦草的联合毒性由拮抗作用转为协同作用。硝磺草酮与铜在水体中赋存可能对水生植物产生潜在的生物安全风险，因此要更加关注水体中不同污染物之间综合效应的防治。     

星星草铁蛋白基因PtFer的克隆及表达分析

目的:从星星草中克隆一个铁蛋白相关基因,分析其序列特征及基因表达模式。方法与结果:构建星星草RACE cDNA文库,根据GenBank中报道的铁蛋白基因EST序列设计引物,利用RACE方法克隆得到星星草铁蛋白基因PtFer的全长cDNA序列(GenBank登录号为HM125047);序列分析表明,PtFer的核苷酸序列长度为751 bp,开放读框为336 bp,编码111个氨基酸残基,预测蛋白质相对分子质量为12.8×103。其蛋白质序列具有铁蛋白的特征性保守区域,与水稻属同一进化分支,与其他禾本科植物铁蛋白的序列相似性达80%以上。Northern杂交分析表明,PtFer的表达量随Na2CO3的浓度和时间的增加而升高,并在12～24 h内持续保持较高的表达水平。结论:用分子生物学及生物信息学技术克隆并分析了星星草铁蛋白基因PtFer的全长cDNA序列及编码蛋白的结构,并初步探索了盐碱胁迫下其表达模式,为研究植物耐盐碱机理奠定了基础。     

螺旋霉素聚酮合成酶基因和抗性基因的克隆与表达的研究

根据不同聚酮合成酶基因DNA的同源性,利用放线紫红素聚酮合成酶基因act Ⅰ,actⅢ作探针,从螺旋霉索产生菌Str．spiramyceticus U-1941基因文库中检测并分离了螺旋霉素聚酮合成酶基因pCN3H8。限制酶酶切分析表明,其分子量为44kb。通过分子杂交实验,将螺旋霉素聚酮缩合酶基因(与act Ⅰ有同源性)及聚酮氧化还原酶基因(与actⅢ有同源性)进行了定位。pCN3H8 DNA在麦迪霉素产生菌变株Str.mycarofaciens sub sp．68中的表达产物,经紫外光谱分析与麦迪霉素相似。pCN3H8在放线紫红素聚酮缩合酶基因缺陷型变株Str．coelicolor TKl7中的表达产物,不具有放线紫红素的色素,其纸层析谱型与螺旋霉素有显著差别。pCN3H8在变青链霉菌Str．lividans TK24中的表达产物,也具有抗菌活性。将pCN3H8 DNA转化对螺旋霉素敏感的Str.griseofuscus原生质体,获得了螺旋霉素抗性的表达。从转化子中分离得到了质粒DNA pSG3,其分子量为7．0kb,可能是pCN3H8DNA转化Str．grlseofuscus时在体内缺失而形成。再转化实验证明,宿主菌对螺旋霉索的抗性,确实是由于pSG3 DNA作用的结果。含质粒pCG4,pSG3的螺旋霉素产生菌Str．Ambofaciens转化子螺旋霉素的产率明显提高。     

丹参2 酮戊二酸依赖性双加氧酶基因克隆及表达分析

以商洛紫花丹参为材料,对其转录组序列SRA020132进行Blast分析,采用PCR技术克隆得到丹参2-酮戊二酸依赖性双加氧酶基因Sm2-ODD1,GenBank登录号为JN935923。Sm2-ODD1基因全长1 365bp,包含3个外显子和2个内含子;cDNA全长1 189bp,读码框951bp,编码316个氨基酸残基;预测的编码蛋白具有2-酮戊二酸依赖性双加氧酶中结合2-酮戊二酸和亚铁离子的"H-T-D"、"H-X"和"R-Y-S"保守基序以及"果冻状"空间结构。表达分析显示,Sm2-ODD1在丹参各个器官都表达,但表达水平具有组织特异性,在根中表达量最高,在叶中表达量最低;该基因表达明显受到MeJA、GA3和ABA的诱导,可能参与了丹参萜类代谢下游途径。     

酮古龙酸菌细胞色素c基因的克隆及表达

目的:从酮古龙酸菌SCB329中分离细胞色素c(Cytc)相关基因,在大肠杆菌中进行表达并验证。方法:根据酮古龙酸菌SCB329基因组序列设计引物,通过PCR从SCB329基因组中扩增cytc基因,酶切后连接pET22b表达载体,转化至大肠杆菌DH5α后提取质粒,经PCR、质粒双酶切及测序鉴定后,转入大肠杆菌BL21(DE3),并对表达条件进行考察;用Chelating Sepharose珠粒对可溶性的Cytc-His融合蛋白进行纯化;经光谱扫描和血红素染色等方法对表达蛋白定性分析。结果:PCR扩增的cytc基因长513 bp;重组菌在IPTG浓度为0.025 mmol/L的条件下,于28℃诱导10 h后,SDS-PAGE分析可见表达条带,相对分子质量约为18×103;Ni柱亲和层析纯化得到目的蛋白,纯化蛋白经光谱扫描呈现Cytc特征峰,血红素染色呈现阳性结果。结论:从酮古龙酸菌SCB329中分离得到一种cytc基因,并表达纯化了融合蛋白Cytc-His,纯化蛋白呈现Cytc特性,为研究酮古龙酸菌中产酸关键酶的电子传递机制奠定了基础。     

发状念珠藻醛酮还原酶基因NfAKR的克隆与表达

以发状念珠藻细胞为试材,采用PCR技术克隆了醛酮还原酶基因的开放阅读框(ORF)序列,命名为NfAKR。对基因序列特征进行了生物信息学分析,根据其编码氨基酸序列预测了NfAKR蛋白的三维结构,同时探讨了PEG-6000胁迫下NfAKR的表达特性。结果表明:NfAKR基因的编码序列长912bp,编码304个氨基酸,预测其编码蛋白的相对分子量为33.51kD,理论等电点为4.94,具有醛酮还原酶超家族保守结构域。NfAKR蛋白主要由10个α-螺旋和11β-折叠组成,中间形成一个疏水穴,作为酶的催化活性中心。NfAKR与点形念珠藻处在同一进化枝上,具有较近的亲缘关系。qRT-PCR分析显示,PEG-6000胁迫下NfAKR基因上调表达,当PEG-6000浓度为8%时,其相对表达量为5.66并达到峰值。依据NfAKR基因响应干旱胁迫上调表达的特性,推测醛酮还原酶可能参与发状念珠藻抵御干旱胁迫过程。     

菠菜乙醇酸氧化酶基因的克隆及表达

采用RT-PCR技术从菠菜总RNA中分离扩增了乙醇酸氧化酶（GO）基因的cDNA序列,首先克隆到质粒pMD18T,进行了测序。然后将乙醇酸氧化酶的cDNA分别亚克隆至质粒pThioHisC、 pTIGTrx、pBV220和pET-2b(+),分别转化大肠杆菌DH5α和BL21（DE3）,并对重组乙醇酸氧化酶在大肠杆菌中的表达进行了研究。SDSPAGE和酶活分析表明,菠菜乙醇酸氧化酶在E.coli BL21 (DE3) （pTIGTrxGO）和E.coli BL21(DE3) （pET-22b(+)GO）里得到了高水平的表达,其中E.coli BL21(DE3) （pET-22b(+)GO）的乙醇酸氧化酶活性较高。     

星星草脱氢抗坏血酸还原酶（PtDHAR）cDNA的克隆及表达分析

脱氢抗坏血酸还原酶是抗坏血酸代谢循环中的关键酶,在多种植物中与抗胁迫相关。为了获得抗盐碱植物星星草中该基因序列,利用RACE技术,从星星草中克隆出脱氢抗坏血酸还原酶基因（PtDHAR）的cDNA全长序列,其GenBank登录号为HM125046。PtDHAR cDNA核苷酸序列长度为987bp,开放阅读框为639bp,编码213个氨基酸。该基因编码的氨基酸序列与水稻、小麦等禾本科作物具有很高的同源性。Northern杂交分析表明,该基因在盐碱胁迫下表达量显著升高。     

Molecular cloning and expression analysis of RrNHX1 and RrVHA-c genes related to salt tolerance in wild Rosa rugosa

Salt stress is one important factor influencing the growth and development of plants, and salt tolerance of plants is a result of combined action of multiple genes and mechanisms. Rosa rugosa is not only an important ornamental plant, but also the natural aromatic plant of high value. Wild R. rugosa which is naturally distributed on the coast and islands of China has a good salt tolerance due to the special living environment. Here, the vacuolar Na⁺/H⁺ reverse transporter gene (NHX1) and the vacuolar H⁺-ATPase subunit C gene (VHA-c) closely related to plant salt tolerance were isolated from wild R. rugosa, and the expression patterns in R. rugosa leaves of the two genes under NaCl stress were determined by real-time quantitative fluorescence PCR. The results showed that the RrNHX1 protein is a constitutive Na⁺/H⁺ reverse transporter, the expression of the RrNHX1 gene first increased and then decreased with the increasing salt concentration, and had a time-controlled effect. The RrVHA-c gene is suggestive of the housekeeping feature, its expression pattern showed a similar variation trend with the RrNHX1 gene under the stress of different concentrations of NaCl, and its temporal expression level under 200 mM NaCl stress presented bimodal change. These findings indicated that RrNHX1 and RrVHA-c genes are closely associated with the salt tolerance trait of wild R. rugosa.     

Discovery of (2-benzoylethen-1-ol)-containing 1,2-benzothiazine derivatives as novel 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibiting-based herbicide lead compounds

A series of (2-benzoylethen-1-ol)-containing benzothiazine derivatives was synthesized, and their herbicidal activities were first evaluated. The bioassay results indicated that some of 3-benzoyl-4-hydroxy-2-methyl-2H-1,2-benzothiazine-1,1-dioxide derivatives displayed good herbicidal activity in greenhouse testing, especially, compound 4w had good pre-emergent herbicidal activities against Brassica campestris, Amaranthus retroflexus and Echinochloa crusgalli even at a dosage of 187.5 g ha⁻¹. More importantly, compound 4w displayed significant inhibitory activity against Arabidopsis thaliana HPPD and was identified as the most potent candidate with IC₅₀ value of 0.48 μM, which is better than the commercial herbicide sulctrione (IC₅₀ = 0.53 μM) and comparable with the commercial herbicide mesotrione (IC₅₀ = 0.25 μM). The structure–activity relationships was studied and provided some useful information for improving herbicidal activity. The present work indicated that (2-benzoylethen-1-ol)-containing 1,2-benzothiazine motif could be a potential lead structure for further development of novel HPPD inhibiting-based herbicides.     

Gene cloning and expression in lactic streptococci

Abstract Recent developments have made the mesophilic lactic streptococci, widely used in dairy fermentations, accessible to genetic manipulation. Several host-vector systems have been described which currently are used in the cloning and expression of homologous and heterologous genes. The essential elements of these systems, the various cloning strategies and the first successful cloning experiments are described with emphasis on the molecular organization of proteinase genes. In addition, the organization and nucleotide sequence of signals which are involved in gene expression in lactic streptococci are summarized.     

Molecular cloning and expression of the genes encoding the Escherichia coli K4 capsular polysaccharide, a fructose-substituted chondroitin

The majority of capsular polysaccharides (K antigens) are linear molecules and their genes have a common functional organisation encoding common steps in capsule biogenesis. However, the K4 antigen is a substituted polymer composed of a chondroitin backbone with a fructose side chain. In order to determine whether K4 biosynthesis uses these common mechanisms the K4 antigen genes were cloned. DNA probes taken from the two conserved regions of the K1 genes were used to isolate one plasmid, pRD1, homologous to both probes. Immunological analysis was used to show that pRD1 directs the production of the substituted K4 antigen on the cell surface. Southern hybridisation was used to show that the cloned genes are organised in the same way as other K antigen gene clusters. We conclude that the branched K4 antigen is handled by the same post-polymerisation mechanisms as other linear K antigens.     

芹菜韧皮部蛋白基因的分离与表达分析

韧皮部蛋白在维持植物形态,物质转运以及植物伤口保护等方面起着重要作用。本研究以地域来源和性状特性差异均较大的两个芹菜品种‘六合黄心芹’和‘美国西芹’为试验材料,利用RT-PCR技术获得这两种芹菜韧皮部蛋白基因的cDNA序列。结果显示：这两种芹菜来源的韧皮部蛋白基因全长均为546 bp,编码181个氨基酸。两者核苷酸序列有3个位点的不同,分别为：88G/A、399T/C和489T/C;在氨基酸序列上有一个位点的不同,为30T/A。预测其蛋白质分子量为19 kD,pI值为9.18。‘六合黄心芹’和‘美国西芹’的韧皮部蛋白与忽地笑等植物的韧皮部蛋白相似度较高,在保守位置分别具有5个亮氨酸残基和4个色氨酸残基。实时定量PCR表达分析表明,该基因主要在芹菜的茎和根等部位表达,具有明显的组织特异性。     

Effect of classic preconditioning on the gene expression pattern of rat hearts: a DNA microarray study

With the aim to enhance the plant vitamin E content, the barley gene encoding 4-hydroxyphenylpyruvate dioxygenase was overexpressed in tobacco plants under control of the 35S promoter. Transgenic lines have a higher capacity for homogentisate biosynthesis as evident by a more than 10-fold higher resistance towards the bleaching herbicide sulcotrione. Seeds from transgenic lines have an up to two-fold enhanced level of vitamin E without a change in the ratio of γ-tocopherol and γ-tocotrienol. While the vitamin E content is not affected in leaves, the level of plastoquinone is enhanced in leaves of transgenic lines during leaf senescence.     

玉米蔗糖磷酸合成酶(SPS)基因的克隆及表达载体的构建

利用RT-PCR方法从玉米幼苗叶片总RNA中克隆出玉米的蔗糖磷酸合成酶(SPS)基因的全长cDNA片段。该片段与文献报道的序列具有99%的同源性。并分别构建了以双CaMV35S为启动子,以Tnos为终止子的植物双元表达载体PBISPS和以Pcab为启动子,以T35S为终止子的植物表达载体PBSPS,其中PBISPS含有NPTⅡ选择标记基因,PBSPS不含选择标记基因。     

高原鼠兔脑红蛋白基因的克隆与组织表达

克隆高原鼠兔脑红蛋白(Neuroglobin,NGB)基因编码区并检测其在成年高原鼠兔脑组织和其他组织中的表达,同时探讨高原鼠兔低氧适应的分子生物学机制。从高原鼠兔脑组织中提取总RNA,通过RT - PCR 获得高原鼠兔NGB cDNA,将其与pGEM - T Easy 载体连接,构建重组质粒,蓝白斑筛选阳性克隆并进行鉴定和测序;制备地高辛标记的RNA 探针并采用原位杂交法(In suit hybridization,ISH) 分析脑红蛋白基因在高原鼠兔脑组织中的分布;采用RT - PCR 和蛋白印记(Western blot)检测高原鼠兔不同组织中脑红蛋白的表达含量。将含有目的片段的阳性克隆经测序和Blast 分析,显示其部分编码序列与GenBank 中绵羊、大鼠等同源性很高(大于84% ),表明本实验所克隆的序列为脑红蛋白基因;原位杂交结果显示NGB 在青藏高原土著动物高原鼠兔脑部分布较为广泛;高原鼠兔不同组织中都有NGB mRNA 表达,NGB 基因并不是中枢神经系统所特有的,睾丸和肾上腺也有较高的表达。NGB 基因在高原鼠兔脑组织和其他组织中分布较为广泛,推测NGB mRNA 可能在高原鼠兔机体较为广泛的区域中发挥着作用,同时为高原低氧适应相关基因的研究提供了实验依据。     

Enhancement of nematicidal potential through cloning and expression of chitinase gene from Bacillus subtilis subsp. Subtilis BTN7A strain

A gene encoding chitinase from B. subtilis has been isolated after optimization of PCR conditions. It was cloned with two different prometers, T7 promoter of the pJET1.2/blunt cloning vector and the SP6 promoter of pGEM®-T Easy vector. After transforming E. coli DH5α, two transformants were selected, CHI-NRC-4 from the first vector and T-CHI-NRC-6 from the second vector, and used for further studies. The complete CDS sequence of chitinase gene was determined and submitted to GenBank with the accession number KX268692.1. Culture supernatants of E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) were investigated for their inhibitory effect on M. javanica egg hatch under laboratory conditions. Result showed up to 96% inhibition in egg hatching due to both E. coli transformants as compared to control which reflect the same expression efficiency of both used prometers. A greenhouse experiment was carried out to evaluate the nematicidal effect of culture supernatants of the two transformts E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) against M. javanica infected eggplant. Obtained results showed a significant reduction in nematode population in soil and roots and enhancement in eggplant growth parameters as compared to control.