生物学自主设计型实验探索——以叶绿体DNA提取为例

指出了分子生物学是生命科学相关学科的主干课程,具有很强的实践性。同时,学生掌握分子生物学的实验方法和技术手段,对培养自身科研能力具有重要意义。结合当前高校对于绿色植物叶绿体DNA提取的实验,从丰富教学方法、更新教学手段、改进考核方式等方面进行了改革与探索。以期提高学生学习质量,强化实验技能及综合能力培养,为高校培养高素质创新型人才奠定基础。     

干制羊肉基因组DNA不同提取方法的比较

为了研究干制加工羊肉基因组DNA的最佳提取方法,本试验采用传统酚-氯仿法、磁珠法、改良CTAB法、离心柱法分别提取干制处理后的羊肉基因组DNA,并对4种方法提取的羊肉基因组DNA浓度、纯度、完整性以及提取所需时间、PCR扩增效果等进行比较。结果表明,采用磁珠法提取DNA的效果更好,DNA浓度为118.87 ng·μL^-1,A₂₆₀/A₂₈₀值为1.89,而且此方法具有提取时间短、效率高、污染小等特点。本研究结果为干制加工羊肉基因组DNA的大批量提取和检测提供了参考依据。     

DNA条形码技术在中药领域的应用

DNA条形码技术可对物种进行快速自动鉴定,具有鉴定准确、结果稳定、操作简便、适用广泛等特点,目前在中药领域应用较多。从DNA条形码技术在中药材鉴定、种植、流通、市场监管、中成药鉴定和药用植物种质资源调查等中药领域的多个方面进行综述,并探讨DNA条形码技术在中药领域的优势和不足,以期为DNA条形码技术在中药领域的研究提供新的思路。     

CUDC-907 induces DNA damage and autophagy in glioma cells

AIM:To investigate the effect of CUDC-907, a dual histone deacetylase (HDAC) and phosphatidylinositol 3-kinase (PI3K) inhibitor, on the DNA damage, cell cycle distribution and autophagy in human glioma U251 cells. METHODS:U251 cells were treated with CUDC-907 of different concentrations, and the cell viability was detected by MTT assay. The quantitative γ-H2AX foci were determined by laser scanning confocal microscopy. The cell cycle distribution of U251 cells was examined by flow cytometry. The protein expression was determined by Western blot analysis. RESULTS:CUDC-907 inhibited the cell viability and the phosphorylation of Akt and p70 ribosomal protein S6 kinase (p70s6K) in the U251 cells (P<0.05). In CUDC-907-treated cells, the number of γ-H2AX foci and protein expression of γ-H2AX were increased significantly (P<0.05). CUDC-907 also induced cell arrest in the G₂/M phase by up-regulating the expression of p21, and inhibiting the protein level of cyclin B1 and the phosphorylation of cell division cycle protein 2 (Cdc2). In addition, CUDC-907 triggered cell autophagy, and inhibition of autophagy increased CUDC-907-induced DNA damage of U251 cells. CONCLUSION:CUDC-907 significantly inhibits PI3K/Akt signaling pathway, induces DNA damage and arrests cell cycle in G₂/M phase. Blockage of autophagy promotes CUDC-907-induced DNA damage of U251 cells.     

转基因玉米TC1507质粒DNA标准物质的研制

标准物质具有特定量值、均匀性和稳定性三大典型特征,也是其作为测量标尺的依据。转基因生物标准物质是我国转基因产品标识制度顺利实施的关键技术支撑之一。文章以转基因玉米TC1507为对象,制备了转化体特异性的新型质粒DNA标准物质pTC1507,并对其均匀性、稳定性、量值进行了评价和测定。测试结果表明,制备的质粒DNA标准物质具有良好的瓶间和瓶内均匀性,pTC1507稳定性可靠,可以在-20 ℃稳定放置6个月以上。经过测序和实时荧光定量PCR定值以及不确定度评估,pTC1507标准物质的量值是1.01±0.053。均匀性、稳定性和量值结果表明,研制的转基因玉米TC1507质粒分子标准物质符合标准物质的典型要求,可以代替传统的基体标准物质应用于转基因玉米检测,解决传统标准物质获取困难、制备复杂、成本高等不足。     

植物DNA甲基化研究进展

植物经常暴露在各种生物和非生物的胁迫之下,这些胁迫会影响植物的生长发育和繁殖并最终导致植物死亡。为了抵御不利的环境条件,植物已经进化出复杂而精细的网络来感知胁迫并激活防御系统。为此,植物激活许多信号转导通路,这些信号转导通路可以改变一些胁迫响应基因的表达,从而引起植物形态、生理和生化的改变以适应逆境。DNA胞嘧啶甲基化是高等真核生物的主要表观遗传机制之一,在维持基因组稳定性和调节基因表达方面起着关键作用。表观遗传变异比遗传变异更为灵活。一旦环境条件发生变化,为了适应新的环境植物都会发生表观遗传的改变。许多研究表明DNA甲基化参与植物的发育和应激反应。基于相关研究对DNA甲基化进行了综述,对植物逆境胁迫有重要意义。     

大鲵幼体蛋白质的需求量

为评定大鲵幼体对饲料蛋白质的需求量,以鱼粉为主要蛋白源配制6种蛋白质水平(干样基础)的实验饲料:D1(43.7%)、D2(47.1%)、D3(51.3%)、D4(55.7%)、D5(59.9%)和D6(64.4%),饲喂初始体质量为(20.99±0.15)g的大鲵幼体92 d。结果显示,①饲料蛋白质水平对大鲵增重率有显著影响,在D4组达到最大值,较D1组增加了276.4%,且全鲵蛋白质沉积率和肌肉RNA、RNA/DNA值、胃蛋白酶、H^+-K^+-ATPase、胰蛋白酶、脂肪酶和Na^+-K^+-ATPase、肝脏超氧化物歧化酶(SOD)均在D4组达到最佳,而肝脏和肠道丙二醛(MDA)在该组均达到最低;②随饲料蛋白质水平增加,肌肉粗蛋白线性增加,全鲵脂肪线性下降,全鲵水分和粗灰分在各组间差异不显著,全鲵粗蛋白先增加后趋于稳定,在D4组达到最大;③大鲵皮肤胶原蛋白含量在D4组达到最高,较D1组增加了27.83%。研究表明,以增重率、肌肉RNA/DNA值、蛋白质沉积率和皮肤胶原蛋白含量为评价指标,通过二次回归方程分析得到大鲵幼体饲料的最适蛋白质水平为55.9%~58.3%(干样基础),该饲料蛋白质水平能显著提高大鲵幼体胃的泌酸能力、机体消化吸收和抗氧化能力,增加鲵体营养素的沉积,从而促进生长和饲料的转化;而低蛋白质水平饲料显著抑制大鲵的生长。     

Plant regeneration via protoplast electrofusion in cassava

Protoplast electrofusion between callus protoplasts of cultivar TMS60444 and mesophyll protoplasts of cultivar SC8 was performed as an approach for the genetic improvement of cassava. The fusion products were subsequently cultured in protoplast culture medium(TM2 G) with gradual dilution for approximately 1–2 months. Then the protoplast-derived compact calli were transferred to suspension culture medium(SH) for suspension culture. The cultured products developed successively into embryos, mature embryos, and shoots on somatic embryo emerging medium(MSN), embryo maturation medium(CMM), and shoot elongation medium(CEM), respectively. And the shoots were then rooted on Murashige and Skoog(1962) medium(MS). Sixty-six cell lines were obtained and 12 of them developed into plantlets. Based on assessment of ploidy level and chromosome counting, four of these plantlets were tetraploid and the remaining eight were diploid. Based on assessment of ploidy level and simple sequence repeat(SSR) analysis, nine tetraploid cell lines, one diploid variant plant line and nine variant cell lines were obtained. The diploid variant plant line and the nine variant cell lines all showed partial loss of genetic material compared to that of the parent TMS60444, based on SSR patterns. These results showed that some new germplasm of cassava were created. In this study, a protocol for protoplast electrofusion was developed and validated. Another important conclusion from this work is the confirmation of a viable protocol for the regeneration of plants from cassava protoplasts. Going forward, we hope to provide technical guidance for cassava tissue culture, and also provide some useful inspiration and reference for further genetic improvement of cassava.     

Effects of different molecular weights of chitosan on methane production and bacterial community structure in vitro

As a new feed additive, chitosan has been shown in recent years to have a certain role in reducing methane emissions from the gastrointestinal tracts of ruminants. However, the effects of chitosan with different molecular weights on rumen fermentation, methane production and bacterial community structure are not yet clear. A basal diet without chitosan served as the control(CTL), and the treatment diets were supplemented with chitosan with different molecular weights: 1 000(1K), 3 000(3K), 5 000(5K), 50 000(5W) and 200 000(20W) dry matter(DM). Six fermentation units per treatment were established. Gas chromatography was used to measure the concentrations of methane, H_2 and volatile fatty acids(VFAs). The bacterial 16S rRNA genes were sequenced with an Illumina MiSeq platform and analysed to reveal the relative abundances of bacterial community taxa. The results showed that the propionate proportion was significantly increased by the addition of chitosan with different molecular weights(P0.05), while methane production and the acetate proportion were significantly decreased(P0.05). The relative abundances of Rikenellaceae_RC9_gut_group and Prevotellaceae_UCG_003 were significantly increased in the 3K chitosan group compared with the CTL group, whereas the relative abundance of Ruminococcaceae_NK4A214_group was significantly decreased(P0.05). Correlation analyses of the relative abundances of the bacterial genera showed that Prevotella was positively related to propionate production(P0.05). In conclusion, 3K chitosan could reduce methane production by replacing fibrolytic bacteria(Firmicutes and Fibrobacteres) with amylolytic bacteria(Bacteroidetes and Proteobacteria) in the bacterial community structure.     

基于DNA条形码的红翅大小蠹和黄杉大小蠹分子鉴定

外来有害生物的快速准确鉴定有助于提高口岸检疫效率。红翅大小蠹(Dendroctonus rufipennis)和黄杉大小蠹(D. pseudotsugae)作为大小蠹属的非中国种是我国口岸原木检疫中频繁截获的检疫性有害昆虫。本研究以mtDNA COI基因5′端和3′端序列为标记对红翅大小蠹与黄杉大小蠹的11个样本开展分子鉴定有效性评价。结果显示,当以COI基因5′端和3′端为靶标时,两种大小蠹间的平均遗传距离分别为0.181和0.144,是种内遗传距离的60.3和48倍,且种内、种间遗传距离间无重叠现象,在系统发育树上都能聚为独立的分支,表明mtDNA COI基因5′端和3′端序列均可有效区分红翅大小蠹与黄杉大小蠹。