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1.
茶树β-1,3-葡聚糖酶基因cDNA片段的克隆与序列分析   总被引：2，自引：0，他引：2  
文章首次从茶树中克隆出-β1,3-葡聚糖酶基因cDNA1369bp连续序列。根据已发表的植物中β-1,3-葡聚糖酶基因的保守序列,设计一对简并引物,采用RT-PCR技术,从茶树中扩增出366bp的cDNA片段。根据此片段序列设计特异引物,利用3'RACE获得-β1,3-葡聚糖酶cDNA 3'端1252bp的cDNA片段。序列分析表明:该基因cDNA 3'端核苷酸序列及其推测的氨基酸序列与其他植物的-β1,3-葡聚糖酶基因家族的cDNA相应序列同源性为50%-90%,经序列拼接,本研究从茶树中克隆出-β1,3-葡聚糖酶基因cDNA3'端1369bp的连续序列,GenBank登录号为AF399920。  相似文献   

2.
根据GenBank发布的基因序列,设计PCR引物,分别从诸葛菜(Orychophragmus violaceus)的花瓣基因组DNA和cDNA克隆到查尔酮合成酶基因,并定名为OvCHS,序列已上传至NCBI数据库,登陆号为EF408918。序列分析表明,OvCHS基因的基因组全长为1263 bp,具一个75 bp的内含子,编码区全长为1188 bp,编码395个氨基酸。与模式植物拟南芥(Arabidopsis thaliana)查尔酮合成酶基因AtCHS比较发现,两基因编码区有135个碱基不同,相似性为88.64%,氨基酸序列中仅16个氨基酸残基的差异,相似性达95.95%。  相似文献   

3.
谷胱甘肽过氧化酶2(GPx2),是生物体内重要的一类抗氧化酶,能够通过酶解过氧化氢阻断活性氧(ROS)的有害作用。本研究从七鳃鳗的血液c DNA文库中克隆了GPx2 c DNA全长和基因组DNA序列。序列分析表明,其c DNA全长为868 bp(Gen Bank序列号KM211710),包括53bp 5′非编码区、251bp 3′非编码区和564 bp开放阅读框,编码由187个氨基酸组成的多肽。基因组DNA序列(Gen Bank序列号KM211711)揭示了基因组特征含有2个外显子和1个内含子结构。系统发育分析表明,七鳃鳗GPx2(Lj GPx2)与其它的GPx2具有很高的同源性。利用实时荧光定量PCR检测到GPx2基因在七鳃鳗各组织中广泛表达,其中在口腔腺、心脏、卵、生殖腺中表达量较高。此外,用脂多糖(LPS)体内刺激七鳃鳗后发现,Lj GPx2 m RNA在生殖腺中表达量显著升高。本研究为探讨GPx2在七鳃鳗的免疫应答中的作用提供了理论依据。  相似文献   

4.
目的:研究与丙型肝炎病毒(HCV)基因RNA序列同源的人染色体外环状DNA(eccDNA)序列.创新点:首次从HCV阴性者eccDNA中检测到HCV 5’-非编码区(5’-NCR)基因组RNA序列,验证了我们的假设: HCV同源DNA序列存在于人的外周血单核细胞的eccDNA组分中.方法:用分离的eccDNA进行HCV特异的聚合酶链反应(PCR),采用核苷酸序列同源性搜索分析软件(BLASTn)对测序结果进行比对分析,并检测其甲基化模式.结论:实验结果证实了我们的假设:即部分HCV 5’-NCR基因组RNA序列存在于外周血单核细胞的eccDNA组分.同时,甲基化分析结果显示了个体间的甲基化模式所代表的受遗传调控的表观遗传特征.  相似文献   

5.
目的:克隆双头菌WH-1环氧乙烷二酸水解酶(ORCH)的基因并研究其酶学性质。创新点:首次获得双头菌ORCH的基因,且该酶催化效率高,热稳定性好。方法:柱层析纯化ORCH后,进行酶学性质研究;通过蛋白末端测序和PCR获得其基因序列;通过二级结构预测和多序列比对进行ORCH序列分析。结论:来源于双头菌WH-1的ORCH是迄今报道的催化效率和热稳定性最好的ORCH,为L(+)-2,3-二羟基丁二酸的生产提供了新的催化剂。  相似文献   

6.
新城疫病毒(NDV)ND-xx08毒株经10 d龄SPF鸡胚增殖后,提取其基因组RNA并反转录成cDNA,用NDV F基因特异性引物,经PCR扩增后获得与F基因预期大小一致的DNA片段。将NDV F基因片段克隆到pMD18-T载体上,并进行EcoR I和Hind III双酶切鉴定和测序鉴定。结果显示,ND-xx08毒株F基因片段的长度为1 662 bp,共编码554个氨基酸,F蛋白的裂解位点为112R-R-Q-K-R-F117,是典型强毒株氨基酸序列结构。将NDV ND-xx08株F基因的47 bp到420 bp序列与新城疫病毒基因型I至基因型Ⅸ毒株的相同序列绘制病毒基因进化树,显示ND-xx08分离株属于基因Ⅶe型。将NDV ND-xx08株F全基因与国内外发表的23株NDV F基因核苷酸序列和氨基酸序列的同源性比较分析,结果表明,其核苷酸序列的同源性在82.7%~97.8%之间,氨基酸同源性在87.5%~97.7%之间。  相似文献   

7.
《大连大学学报》2016,(3):56-61
丝氨酸乙酰转移酶(SAT,EC 2.3.1.30)是硫同化中非常重要的一个酶,是半胱氨酸合成的关键酶,催化丝氨酸转化为氧乙酰丝氨酸的反应,后者再生成半胱氨酸。本研究从红藻门杉藻科角叉菜(Chondrus crispus)c DNA文库中克隆了角叉菜丝氨酸乙酰转移酶基因(cys E)全长序列(Gen Bank序列号KT963952)。序列分析表明,其ORF区长1143 bp,编码由380个氨基酸组成的蛋白,理论分子量为40.60 k Da,理论等电点为6.26。生物信息学分析表明角叉菜SAT与其他物种的SAT的氨基酸序列具有较高的同源性。本研究为探讨SAT在角叉菜硫代谢中的作用提供了实验基础。  相似文献   

8.
芪合酶是白藜芦醇合成的关键酶。本文分别以传统CTAB法、传统SDS法和改良SDS法对虎杖基因组DNA进行提取,获得符合分子生物学试验要求的高质量DNA。利用芪合酶基因保守区段的特异引物进行PCR扩增,将获得的片段进行测序分析,长度为809bp,成功克隆出虎杖芪合酶基因的保守区段,为芪合酶基因工程奠定基础。  相似文献   

9.
采用高保真PCR从我国钩体黄疸出血群赖株基因组中扩增全长mviN基因片段,对钩体黄疸出血群赖株mviN基因进行克隆.结果显示,所克隆的mviN基因的核苷酸和氨基酸序列与已发表的mviN基因序列(Accesion No.in GenBank: NC004343)同源性分别为99.68%和99.42%.因此可以认为,我们成功克隆了mviN基因,并为该基因的后续研究奠定了基础.  相似文献   

10.
珙桐rbcL基因的克隆与序列分析   总被引：1，自引：0，他引：1  
根据已知植物rbcL基因设计引物,以珙桐叶片总DNA为模板,PCR扩增目的DNA片段,克隆入pDM19-T载体.阳性克隆鉴定后进行测序,序列分析结果表明:该基因片断长为1266 bp,包括1031 bp的编码区序列,编码343个氨基酸;其编码区氨基酸与烟草、菠菜、玉米、甘薯、拟南芥、水稻、葡萄、地钱和葡萄柚等9个物种的同源性94.13%以上,并构建了它们间的亲缘关系树.该基因片断的预测晶体结构与菠菜的最相近,推测Lys86、Lys60、Lys179等残基对酶的活性影响较大.  相似文献   

11.
Anoectochilus formosanus, commonly known as “Jewel Orchid”, is a Chinese folk medicine used to treat hypertension, diabetes, and heart disease. The existence of A. formosanus is currently threatened by habitat loss, human and animal consumption, etc. The highly potent medicinal activity of A. formosanus is due to its secondary metabolites, especially kinsenosides and flavonoids. This orchid also has a unique mycorrhizal relationship. Most adult orchids rely on endophytes for mineral nutrition and have complex interactions with them, which are related to plant growth, yield and changes in secondary metabolites. This study investigated the promoting role of F-23 fungus (genus Mycena) on the biomass and contents of kinsenosides and flavonoids of A. formosanus in pot culture. The following were observed after 10 weeks of symbiotic cultivation: increased shoot height, shoot dry weight, and leaf numbers by 16.6%, 31.3%, and 22.5%, respectively; increased contents of kinsenosides, isorhamnetin-3-O-β-d-rutinoside, and isorhamnetin-3-O-β-d-glucopyranoside by 85.5%, 226.1%, and 196.0%, respectively; some hyphae in epidermal cells dyed red and/or reddish brown by safranine; and, significantly reduced number of starch grains in cortical cells. Moreover, F-23 fungus significantly improved the kinsenoside and flavonoid contents of A. formosanus. These findings supported the reports that endophytes can alter the production of secondary metabolites in their plant hosts, although further physiological, genetic and ecological analyses are warranted.  相似文献   

12.
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13.
The heat shock cognate protein 70 (Hsc70) is a member of a 70-kDa heat shock protein (HSP70) family that functions as molecular chaperones. In this study, a novel Hsc70 gene from Chinese soft-shelled turtle (Pelodiscus sinensis) (tHsc70) was identified. The tHsc70 full-length complementary DNA (cDNA) is 2 272 bp long with a 1 941-bp open reading frame (ORF) encoding 646 amino acids. Three characteristic signature regions of the HSP70 family, two major domains of an adenosine triphosphate (ATP)/guanosine triphosphate (GTP) binding domain (ABD), and a substrate-binding domain (SBD) were present in the predicted tHsc70 amino acid sequence. The tHsc70 gene was expressed in Escherichia coli BL21 and the expression product reacted with the anti-Hsc70 mouse monoclonal antibody by Western blotting. Homology analysis revealed that tHsc70 shared identity from 53.9% to 87.7% at the nucleotide level, and 49.1% to 99.5% at the amino acid level with the known Hsc70s. Phylogenetic analysis showed that tHsc70 was clustered together with the Hsc70 gene of another reptile species (Alligator mississippiensis). The tHsc70 was expressed in the liver, lung, heart, and skeletal muscle. The expression patterns of tHsc70 messenger RNA (mRNA) differed among different tissues under different durations of heat stress at 40 °C. Adaptation at 25 °C for 1 h after heat stress was also different among tissues and length of heat stress. Irrespective of different profiles of expression under heat stress, tHsc70 may play roles in protecting turtles from thermal stress.  相似文献   

14.
Objective: Labisia pumila var. alata, commonly known as ’Kacip Fatimah’ or ’Selusuh Fatimah’ in Southeast Asia, is traditionally used by members of the Malay community because of its post-partum medicinal properties. Its various pharmaceutical applications cause an excessive harvesting and lead to serious shortage in natural habitat. Thus, this in vitro propagation study investigated the effects of different plant growth regulators (PGRs) on in vitro leaf and stem explants of L. pumila. Methods: The capabilities of callus, shoot, and root formation were evaluated by culturing both explants on Murashige and Skoog (MS) medium supplemented with various PGRs at the concentrations of 0, 1, 3, 5, and 7 mg/L. Results: Medium supplemented with 3 mg/L indole-3-butyric acid (IBA) showed the optimal callogenesis from both leaf and stem explants with (72.34±19.55)% and (70.40±14.14)% efficacy, respectively. IBA was also found to be the most efficient PGR for root induction. A total of (50.00±7.07)% and (77.78±16.47)% of root formation were obtained from the in vitro stem and leaf explants after being cultured for (26.5±5.0) and (30.0±8.5) d in the medium supplemented with 1 and 3 mg/L of IBA, respectively. Shoot formation was only observed in stem explant, with the maximum percentage of formation ((100.00±0.00)%) that was obtained in 1 mg/L zeatin after (11.0±2.8) d of culture. Conclusions: Callus, roots, and shoots can be induced from in vitro leaf and stem explants of L. pumila through the manipulation of types and concentrations of PGRs.  相似文献   

15.
In the present study, we investigated the possible toxicity mechanism of lipopolysaccharide (LPS) extracted from Gram-negative bacteria in Eriocheir sinensis hemocytes. Apoptotic hemocytes and reactive oxygen species (ROS) production induced by the LPS were monitored by the combination of flow cytometry and microscope observation. It was shown that LPS induced serious damage on the DNA and morphological changes in hemocytes, including cell shrinkage, fracture of nucleus membrane, margination, condensation and fragmentation of chromatin, and formation of apoptotic bodies indicating obvious hemocyte apoptosis. As compared with the control group, the apoptotic cell ratio increased to 30.61% and 39.01% after 1-h exposure and 57.72% and 75.01% after 2-h exposure to 1 and 10 μg/ml LPS, respectively (P<0.05). Significant outburst of ROS production was observed in LPS-treated hemocytes with approximately 176.6% of relative dichlorofluorescein mean fluorescence at 1-h exposure, followed by a drastic decline (P<0.05). These results indicated that LPS would induce oxidative stress on hemocytes from E. sinensis and cause ROS burst, DNA damage, and subsequently apoptosis. The process of ROS-mediated apoptosis might be one of the potential toxicity mechanisms of LPS on crustacean hemocytes.  相似文献   

16.
Endophytic flora plays a vital role in the colonization and survival of host plants, especially in harsh environments, such as arid regions. This flora may, however, contain pathogenic species responsible for various troublesome host diseases. The present study is aimed at investigating the diversity of both cultivable and non-cultivable endophytic fungal floras in the internal tissues (roots and leaves) of Tunisian date palm trees (Phoenix dactylifera). Accordingly, 13 isolates from both root and leaf samples, exhibiting distinct colony morphology, were selected from potato dextrose agar (PDA) medium and identified by a sequence match search wherein their 18S–28S internal transcribed spacer (ITS) sequences were compared to those available in public databases. These findings revealed that the cultivable root and leaf isolates fell into two groups, namely Nectriaceae and Pleosporaceae. Additionally, total DNA from palm roots and leaves was further extracted and ITS fragments were amplified. Restriction fragment length polymorphism (RFLP) analysis of the ITS from 200 fungal clones (leaves: 100; roots: 100) using HaeIII restriction enzyme revealed 13 distinct patterns that were further sequenced and led to the identification of Alternaria, Cladosporium, Davidiella (Cladosporium teleomorph), Pythium, Curvularia, and uncharacterized fungal endophytes. Both approaches confirmed that while the roots were predominantly colonized by Fusaria (members of the Nectriaceae family), the leaves were essentially colonized by Alternaria (members of the Pleosporaceae family). Overall, the findings of the present study constitute, to the authors’ knowledge, the first extensive report on the diversity of endophytic fungal flora associated with date palm trees (P. dactylifera).  相似文献   

17.
Insect resistance and glyphosate tolerance have been two of the most important traits in the genetic improvement of various crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, Cry1Ac and Cry1Ig, and a modified glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (G10) were combined into a single transferred DNA (T-DNA) fragment and introduced into rice by Agrobacterium-mediated transformation. A transgenic line with single-copy T-DNA insertion named GAI-14 was found to be highly resistant to striped stem borer and rice leaf roller, and tolerant to glyphosate. Analysis of T-DNA border sequence suggested that the transgenes were inserted at the chromosome 3 and appeared to have not interrupted any known or putative genes. A field trial observed no significant difference in the basic agronomic traits between GAI-14 and the recipient rice.  相似文献   

18.
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19.
Lipophilic tea polyphenols (LTP) were prepared by catalytic esterification of green tea polyphenols (GTP) with hexadecanoyl chloride. A novel long-chain acyl-derivative of epigallocatechin-3-o-gallate (EGCG) was first isolated from purification of LTP by high-speed countercurrent chromatography (HSCCC) using a solvent system composed of n-hexane-ethyl acetate-methanol-water (1∶1∶1∶1, v/v). The molecular structure of the acyl-derivative, Epigallocatechin-3-O-gallate-4′-O-hexadecanate, was elucidated by means of elemental analysis, IR,1H-NMR and MS spectra. Project (No. 200010) supported by Zhejiang Provincial Natural Science Foundation, China  相似文献   

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