circRNA-Zfp609对C2C12成肌细胞增殖和分化的影响

【目的】试验旨在探究circRNA-Zfp609调节C2C12成肌细胞增殖和分化的潜在分子机制。【方法】利用RT-PCR和测序分析小鼠骨骼肌组织和C2C12成肌细胞中circRNA-Zfp609的表达,实时荧光定量PCR检测小鼠心脏、肝脏、脾脏、肺脏、肾脏、胃、小肠、骨骼肌组织及增殖12、24、36、48 h和分化0、1、3、5 d的C2C12成肌细胞中circRNA-Zfp609的相对表达量;实时荧光定量PCR检测分化0、1、3、5 d的C2C12成肌细胞中肌细胞生成素（MyoG）和肌球蛋白重链（MyHC）的相对表达量。用circRNA-Zfp609的干扰表达载体（siRNA）干扰细胞,通过CCK-8测定siRNA对C2C12成肌细胞增殖率的影响;用实时荧光定量PCR检测siRNA干扰对circRNA-Zfp609、MyoG和MyHC相对表达量的影响。通过TargetScan 7.0和miRDB软件预测circRNA-Zfp609上与肌肉分化相关的miRNA位点,将筛选的miRNAs的过表达载体转染HEK293T细胞,利用双荧光素酶报告试验验证circRNA-Zfp609与miRNA的互作关系。根据miRNAs对circRNA-Zfp609的互作,构建circRNA-Zfp609的野生型和突变型载体并转染HEK293T细胞,利用双荧光素酶报告试验验证circRNA-Zfp609对miRNA的靶向关系。【结果】PCR和测序结果表明,小鼠骨骼肌中可表达circRNA-Zfp609;circRNA-Zfp609在小鼠骨骼肌中表达水平最高,在其他组织中的表达量由高到低依次是肾脏、肺脏、心脏、肝脏、胃、脾脏和小肠。与12 h相比,在C2C12成肌细胞增殖的36和48 h circRNA-Zfp609的相对表达量显著增加（P<0.05）;与分化第0天相比,在C2C12成肌细胞分化的第1、3和5天circRNA-Zfp609的相对表达量均显著增加（P<0.05）,MyoG、MyHC的相对表达量均极显著增加（P<0.01）。与NC组相比,siRNA组C2C12成肌细胞的增殖率和circRNA-Zfp609相对表达量均极显著降低（P<0.01）,MyoG和MyHC的相对表达量显著降低（P<0.05）。circRNA-Zfp609上有miR-150-5p、miR-327、miR-344g-3p和miR-615-5p 4种与肌肉分化相关的miRNAs。circRNA-Zfp609与miR-615-5p的吸附能力最强,具有靶向结合作用。circRNA-Zfp609可以作为分子海绵与调控肌肉分化相关的miR-615-5p相互作用。【结论】circRNA-Zfp609在小鼠的组织中广泛表达,在骨骼肌中表达水平最高;circRNA-Zfp609在C2C12成肌细胞增殖和分化的不同时期差异表达,circRNA-Zfp609上有4个与肌肉分化相关的miRNAs,其中miR-615-5p与circRNA-Zfp609具有靶向关系。本研究结果可为与家畜骨骼肌生长发育相关的研究提供参考。     

牦牛源BVDV非结构蛋白NS5A的原核表达及抗原表位分析

【目的】利用原核表达系统体外表达牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)NS5A基因,获得非结构蛋白NS5A,对其进行核苷酸、氨基酸序列分析,以解析BVDV非结构蛋白NS5A的功能。【方法】参考BVDV-1型毒株V006的NS5A基因序列(GenBank登录号:KX170647)设计并合成1对特异性引物,以分离到的牦牛BVDV GSTZ毒株cDNA为模板,PCR扩增NS5A基因片段,并克隆至表达载体pET-28a (+)中,构建重组原核表达载体pET28a-NS5A。经酶切初步鉴定及测序鉴定正确后,转化大肠杆菌BL21(DE3)感受态细胞,然后利用IPTG诱导表达。经10% SDS-PAGE电泳及Western blotting分析鉴定重组蛋白的表达,并根据NS5A基因的序列构建遗传发育进化树,利用DNAStar软件预测NS5A蛋白的亲水性、表面可塑性和抗原性等特性,并结合二级结构的预测对NS5A蛋白的B细胞抗原表位进行预测。【结果】PCR扩增NS5A目的基因片段为1 488 bp,双酶切和测序鉴定结果证明,重组质粒pET28a-NS5A构建成功。经10% SDS-PAGE电泳及Western blotting鉴定重组蛋白,表达出了大小为55 ku的目的蛋白,大小与预期结果相符。通过对不同BVDV毒株NS5A基因序列构建遗传发育进化树,显示GSTZ毒株NS5A在遗传进化特征上属于BVDV-1型。NS5A蛋白的亲水性主要位于12—21、32—69、75—113、120—135、143—147、152—163、165—180、215—230、265—274、296—340、348—378、389—447、455—463、469—495位氨基酸处,表面可塑性主要位于14—18、37—42、76—81、86—109、154—160、169—178、218—228、297—309、348—358、365—373、414—442、430—437、454—460位氨基酸处,柔性区域较多,主要位于14—21、37—43、67—82、86—93、97—110、152—158、169—179、218—231、240—255、296—310、313—328、344—359、364—373、413—422和472—483位氨基酸处。NS5A蛋白的B细胞抗原表位主要位于15—18、76—81、154—158、169—178、218—228、297—309、348—358、365—373和414—422位氨基酸处。【结论】成功表达并鉴定了牦牛源BVDV的非结构蛋白NS5A,系统发育进化树表明BVDV GSTZ株基因型属于BVDV-1型,NS5A蛋白具有良好的抗原性,为深入解析BVDV非结构蛋白NS5A的自身结构功能、免疫学特性以及进一步研究非结构蛋白对病毒复制的影响提供参考。     

飞机喷洒阿维灭幼脲防治马尾松毛虫试验

2004年6～7月,运用Y-5B型飞机超低量喷洒25%阿维灭幼脲新型生物复合剂防治闽北林区马尾松毛虫中、重度危害面积1 96万hm2。结果表明,25%阿维灭幼脲是一种高效、稳定、安全、持效期长的生物农药,使用525g·hm-2浓度,飞机防治作业20d后,效果可达90%以上,防治效果好。     

SPOT5卫星遥感信息在森林资源调查中的应用现状与解决思路

介绍利用SPOT5卫星遥感在云南省森林资源调查中的应用情况,总结所取得的成果经验。分析存在的包括对遥感的内涵认识不足、特殊地类不易分辨、树种组成难判读、蓄积量不能直接判读、龄组和郁闭度判读因子粗放等问题,提出了解决问题的思路和建议。     

森林资源调查中Spot5卫星影像的处理方法

利用Spot5卫星进行森林资源调查时,首先要对遥感图像进行处理。文章介绍了处理环境对硬件和软件的要求。其处理过程包括影像配准、分辨率融合、影像拼接、色彩增强、分幅输出等。对其中的一些问题提出了个人见解。     

华北落叶松抚育间伐技术的研究

本文论述了如何以效应生长量分析为基础,通过对华北落叶松人工林间伐林分的研究,建立与间伐有关的系列断面积收获预估模型。以此为基础,提出了一套确定抚育间伐强度和预测目标径级收获量的具体方法.通过对伐前密度为5550株·hm~(-2),地位指数级为10的华北落叶松人工林的研究,提出了间伐起始期15a,间隔期5a,采取4次间伐,间伐株强分别为30.3％,17.1％.22.4％,7.8％的间伐体制。     

The effect of strain, growth stage, and cultivating condition of Ganoderma lucidum on 5α-reductase inhibition

The inhibitory effects of 102 methanol extracts of 40 mycelia, 9 culture fluids, and 53 fruiting bodies of 40 strains of Ganoderma lucidum on 5-reductase were investigated. The methanol extract of the fruiting body of each strain was found to show the strongest 5-reductase inhibitory activity among the extracts tested.     

Reaction behavior of lignin in supercritical methanol as studied with lignin model compounds

 The reaction behavior and kinetics of lignin model compounds were studied in supercritical methanol with a batch-type supercritical biomass conversion system. Guaiacol, veratrole, 2,6-dimethoxyphenol, and 1,2,3-trimethoxybenzene were used as model compounds for aromatic rings in lignin. In addition, 5-5, β-1, β-O-4, and α-O-4 types of dimeric lignin model compounds were used as representatives of linkages in lignin. As a result, aromatic rings and 5-5 (biphenyl)-type structures were stable in supercritical methanol, and the β-1 linkage was not cleaved in the β-1-type structure but converted rapidly to stilbene. On the other hand, β-ether and α-ether linkages of β-O-4 and α-O-4 lignin model compounds were cleaved rapidly, and these compounds decomposed to some monomeric compounds. Phenolic compounds were found to be more reactive than nonphenolic compounds. These results indicate that cleavages of ether linkages mainly contribute to the depolymerization of lignin, whereas condensed linkages such as the 5-5 and β-1 types are not cleaved in supercritical methanol. Therefore, it is suggested that the supercritical methanol treatment effectively depolymerizes lignin into the lower-molecular-weight products as a methanol-soluble portion mainly by cleavage of the β-ether structure, which is the dominant linkage in lignin. Received: December 19, 2001 / Accepted: April 30, 2002 Acknowledgments This research has been done under the research program for the development of technologies for establishing an ecosystem based on recycling in rural villages for the twenty-first century from the Ministry of Agriculture, Forestry and Fisheries, Japan; by a Grant-in-Aid for Scientific Research (B)(2) (no.12460144, 2001.4–2003.3) from the Ministry of Education, Culture, Sports, Science and Technology, Japan; and under the research program from Kansai Research Foundation for Technology Promotion, Japan. The authors thank them for their financial support. This study was presented in part at the 45th Lignin Symposium, Ehime, Japan, October 2000 and the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, Japan, April 2002 Correspondence to:S. Saka     

Determination of nitrobenzene oxidation products by GC and1H-NMR spectroscopy using 5-iodovanillin as a new internal standard

The nitrobenzene oxidation method was modified to obtain more reproducible data and more structural information about lignin, not only by gas chromatography (GC) but also by proton nuclear magnetic resonance (¹H-NMR) spectroscopy for quantitative determination of the oxidation products and to simplify the procedures. The nitrobenzene oxidation mixture was directly extracted after acidification without preextraction of by-products. The direct extraction made the extractive step easy and gave reproducible data. 5-Iodovanillin was selected as a new internal standard. The reason for this selection was that 5-iodovanillin did not exist in the nitrobenzene oxidation products from any plant species and had an aldehyde group whose peak did not overlap with the other aldehyde peaks on an¹H-NMR spectrum. Thus, the use of 5-iodovanillin enabled us to quantifyp-hydroxybenzaldehyde, vanillin, and syringaldehyde in oxidation products on the basis of¹H-NMR analysis as well as GC. Furthermore, more information about the condensed structure of lignin was derived by comparing the¹H-NMR and GC analyses.Part of this work was presented at the 42nd Annual Meeting of the Lignin Symposium, Sapporo, October 1997     

木质素合成酶基因F5H的RT-PCR引物筛选

据EMBL核酸序列库中发表的白杨杂种F5H的cDNA序列,利用Primer 5.0软件设计3组扩增引物。通过PCR及RT-PCR扩增反应,筛选出一组比较合适的引物,确定该引物对的最适反应体系和程序参数。利用该引物对,从正在分化的2年生欧美杨107茎的次生木质部提取的mRNA中RT-PCR扩增出一个867bp的木质素合成特异表达的f5h基因片段。将其克隆到pGM-T载体上,构建了f5h在大肠杆菌中的表达载体,并对引物筛选进行讨论。