荧光实时定量PCR技术及其在水产养殖研究中的应用

荧光实时定量PCR技术是近年来快速发展起来的一门新技术,它是核酸探针技术、荧光共振能量传递技术(Fluorescence Resonance Energy Transfer,FRET)和PCR技术的有机结合。与常规PCR相比,它具有特异性更强、有效解决PCR污染问题、自动化程度高、能较准确定量等特点。本文综述了荧光实时定量PCR技术的基本原理及几种广泛应用的荧光化学方法,并概述了荧光实时定量PCR技术在水产养殖研究领域的应用现状,并对荧光实时定量PCR技术的应用前景进行了展望。     

双流道泵内非定常固液两相流的数值分析

采用Mixture多相流模型、标准的k-ε湍流模型与SIMPLEC算法,应用FLUENT软件对双流道泵内固液两相湍流进行了非定常数值模拟.研究结果表明:不同时刻,泵流道内的压力分布明显不同,呈现周期性变化规律;在叶轮流道内,固相体积浓度分布极不均匀,颗粒主要集中于叶轮出口处的工作面和后盖板上,而在蜗壳流道内,固相颗粒多集中于离叶轮半径较大的蜗壳壁面上.     

汽车前照灯智能控制系统的设计

基于汽车驾驶的安全性,结合传感器技术和微控制技术,对汽车前照灯设计了一套智能控制系统。该系统能在不同的情况下,对前照灯的照射角度和亮度进行自动实时调整和自动选择适宜的灯光模式,实现汽车前照灯的智能化控制,提高了汽车驾驶的安全性、舒适性以及智能感觉。     

Comparative proteomics analysis of maize (Zea mays) leaves infected by small brown planthopper (Laodelphax striatellus)

Maize rough dwarf disease (MRDD) is a viral disease caused by brown planthopper infestation, and leads to great yield loss, especially in China. Comparative proteomics was performed using maize inbred line Zheng 58 and LN 287. MRDD pathogen was detected as rice black-streaked dwarf virus (RBSDV) by quantitative real time PCR (qRT-PCR) in Shandong Province, China. The modified trichloroacetic acid (TCA)/acetone method was used for soluble protein extraction from leaves. Two-dimensional electrophoresis (2-DE) analysis was performed on 24-cm long, pH 4–7 linear immobilized pH gradient (IPG) strips, and gels were stained with silver and coomassie brilliant blue. We identified 944 proteins expressed in RBSDV infected maize leaves by proteomics approaches. Among these, 44 protein spots that revealed a 1.5-fold difference in intensity were identified by mass spectrometry between mock-inoculated and RBSDV infected samples. Among these, 17 and 26 spots were up-regulated, and 27 and 18 spots were down-regulated in the virus infected samples of Zheng 58 and LN 287, respectively. Differential protein spots were analyzed by mass spectrometry identification, which could be divided into six categories. Furthermore, the expression of stress-related proteins was detected and confirmed by qRT-PCR. This study lays the foundation for further investigations, enabling the enhancement of MRDD resistance in maize.     

富含多糖甜玉米幼穗RNA提取及高温胁迫基因表达分析

以改良Trizol试剂法从富含多糖的甜玉米幼穗中提取高质量、完整的总RNA,利用实时荧光定量PCR对高温胁迫下粤甜13雌穗发育的8个差异表达基因进行分析。结果表明,8个基因分为上调表达和下调表达,实时荧光定量PCR和测序法基因表达谱检测的基因上调或下调表达的趋势一致,上调表达基因ZM2G058057和下调表达基因ZM2G059964、ZM2G044670相对表达量较高,可能在高温胁迫影响甜玉米幼雌穗发育过程中起更重要的作用。     

小麦籽粒淀粉合成酶基因表达与酶活性特征的研究

为研究小麦籽粒淀粉合成酶基因表达与酶活性的特征,以普通小麦品种秦麦11(粒重36.942 mg/粒,淀粉含量61.02%)和中优9507(粒重50.636 mg/粒,淀粉含量68.36%)为材料,采用实时荧光定量RT-PCR方法,对灌浆期籽粒淀粉合成酶相关基因的表达进行了研究,并对其表达量与相应酶活性的相关性做了分析.结果表明,腺苷二磷酸葡萄糖焦磷酸化酶基因(AGPase)、束缚态淀粉合成酶基因(GBSSI)、可溶性淀粉合成酶基因(SSS)、淀粉分支酶基因(SBE)表达均呈单峰曲线变化,在花后3 d 内检测不到其表达,花后4～6 d 这些基因开始表达.AGPase和SSS在花后12 d左右表达量达到高峰,GBSSI和SBE在花后15 d左右的表达量最大.自花后18 d开始,各基因的表达量均显著下降.中优9507在表达高峰期(即花后第12、15、18 d)的酶基因相对表达量均显著高于秦麦11,且差异均达显著水平.整个灌浆期间中优9507的四种淀粉合成酶活性高于秦麦11,尤其在灌浆中期差异更显著.相关分析表明,AGPase、SSS、SBE基因在花后15 d的相对表达量与相应酶活性间呈极显著正相关,而GBSS基因的相对表达量与GBSS酶活性间相关不显著,暗示AGPase、SSS、SBE基因在籽粒淀粉合成过程中属于转录水平调控,而GBSSI基因属于转录后调控.     

鱼糜制品中基因组DNA提取方法的比较

将分别采用普通酚-氯仿抽提法和试剂盒(柱吸附法)提取DNA的两种方法加以比较,以确定最适提取鱼糜制品DNA的方法.将鱼肉和鱼糜制品作为原料,用两种方法提取基因组DNA,利用分光光度计测定其A260与A280的吸光值,计算比率估计核酸的纯度,并利用琼脂糖凝胶电泳观察DNA片段在凝胶中的位置;并用线粒体16S rRNA基因PCR扩增产物鉴定所提取的基因组DNA.     

耕作方式对华北冬小麦.夏玉米周年产量和水分利用的影响

在冬小麦季设置秸秆不还田翻耕(CT)、秸秆还田翻耕(CTS)、秸秆还田旋耕(RTS)和免耕秸秆覆盖(NTS)4种处理,研究耕作方式对华北小麦-玉米两熟区作物周年产量和水分利用的影响。结果表明:耕作方式对当季冬小麦产量和水分利用影响显著,对夏玉米产量和水分利用影响不大,但秸秆还田提高了夏玉米产量。RTS、CTS、CT 3个处理小麦季产量差异不显著,而NTS由于有效穗数不足,产量显著低于其他处理;与CT相比,NTS周年产量平均减产5.13%,RTS增产2.69%,CTS增产2.33%。耕作方式对当季小麦土壤水分含量影响大,而对后茬夏玉米土壤水分含量的影响较小。NTS提高了小麦季土壤水分含量,增加了土壤储水量,与CT相比,0~60 cm土壤储水量2010年和2011年分别增加39.07 mm和26.65 mm。从耗水构成来看,土壤水在冬小麦耗水中所占比例最大,其次为灌水和降水;而夏玉米耗水以降水为主,且降水中有一部分转化为土壤水储存起来。NTS提高了冬小麦季土壤储水量,降低了土壤水分的消耗,冬小麦季耗水最少。与CT相比,NTS小麦季平均节水22.40 mm,周年耗水量也以NTS最少;但NTS冬小麦产量降低导致其小麦季和周年水分利用效率均最低。从作物周年产量和水分利用的角度来看,如何提高免耕秸秆覆盖小麦季产量,进而提高周年产量,发挥其节水优势,是该耕作模式在华北地区冬小麦?夏玉米两熟区推广应用亟需解决的关键问题。     

均匀设计优化毛竹EST-SSR PCR反应体系

本研究以毛竹叶片DNA为模板,利用均匀设计U10（54）表,对毛竹EST-SSRPCR反应体系的TaqDNA聚合酶、Mg2＋、dNTPs及引物浓度4因素5个水平进行优化筛选。优化实验结果表明,毛竹EST-SSRPCR的25μL优化反应体系为：10×PCRBuffer（含Mg2＋）2.5μL、TaqDNA聚合酶1.5U,Mg2＋、dNTPs及引物的最适浓度分别是1.0mmol/L、0.1mmol/L、0.48μmol/L;通过梯度PCR试验筛选得到相应引物的最佳退火温度为52.4℃。对优化出的EST-SSRPCR反应体系进行稳定性检测,结果均能获得丰富、稳定、清晰可辨的DNA谱带。该优化体系为利用EST-SSR分子标记对毛竹进行遗传多样性等相关研究工作提供了基础条件。     

Molecular characterization and PCR detection of a nitrogen-fixing Pseudomonas strain promoting rice growth

Nitrogen-fixing plant growth-promoting rhizobacteria (PGPR) from the genus Pseudomonas have received little attention so far. In the present study, a nitrogen-fixing phytohormone-producing bacterial isolate from kallar grass (strain K1) was identified as Pseudomonas sp. by rrs (16S ribosomal RNA gene) sequence analysis. rrs identity level was high with an uncharacterized marine bacterium (99%), Pseudomonas sp. PCP2 (98%), uncultured bacteria (98%), and Pseudomonas alcaligenes (97%). Partial nifH gene amplified from strain K1 showed 93% and 91% sequence similarities to those of Azotobacter chroococcum and Pseudomonas stutzeri, respectively. The effect of Pseudomonas strain K1 on rice varieties Super Basmati and Basmati 385 was compared with those of three non-Pseudomonas nitrogen-fixing PGPR (Azospirillum brasilense strain Wb3, Azospirillum lipoferum strain N4 and Zoogloea strain Ky1) used as single-strain inoculants. Pseudomonas sp. K1 was detected in the rhizosphere of inoculated plants by enrichment culture in nitrogen-free growth medium, which was followed by observation under the microscope as well as by PCR using a rrs-specific primer. For both rice varieties, an increase in shoot biomass and/or grain yield over that of noninoculated control plants was recorded in each inoculated treatment. The effect of Pseudomonas strain K1 on grain yield was comparable to those of A. brasilense Wb3 and Zoogloea sp. Ky1 for both rice varieties. These results show that nitrogen-fixing pseudomonads deserve attention as potential PGPR inoculants for rice.