噬菌体DNA的快速抽提

介绍一种噬菌体DNA的快速抽提方法.用聚乙二醇沉淀噬菌体颗粒,然后经DEAE纤维素纯化处理和酚抽提.与传统的噬菌体DNA纯化方法相比,改进后的方法方便、快速、经济,可获得高纯度的噬菌体DNA.     

一种新的以细胞表面受体为靶向的基因导入系统

鉴于胰岛素样生长因子Ⅰ号及Ⅱ号的受体 (IGFⅠR ,IGFⅡR)在人原发性肝癌中过量表达 ,以及表皮细胞生长因子受体 (EGFR)在多种人恶性肿瘤过量表达 ,设计和合成了针对IGFⅠR及IGFⅡR的 1 4肽E5和针对EGFR的 1 6肽GE7,以及流感病毒血凝素功能域 2 0肽HA2 0作为内吞小体释放寡肽 (Endosomereleasingoligopeptide,EOP) ,将它们分别与多聚阳离子多肽 (Polycationic polypeptide ,PCP)———多聚赖氨酸 (Polylysine ,PL)或鱼精蛋白 (Protamine,PA)共价连接 ,藉静电效应与DNA形成一个复合体 (E5 PCP/DNA/PCP HA2 0 ,GE7 PCP/DNA/PCP HA2 0 ) ,即构建的新的受体介导的靶向性非病毒型基因导入系统 .体内、外实验结果表明它们相对靶向且高效地将外源基因导入人恶性肿瘤细胞并得到预期表达     

基于DUS测试的上海粳稻地方品种遗传多样性分析

利用水稻DUS测试指南(2012版)中的38个水稻标准品种和52个基本测试性状对上海地区的172个粳稻地方品种进行遗传多样性分析,并与14个上海地区选育的申请品种保护的粳稻品种作比较。结果表明:地方品种表型性状共检测到176个等位变异,平均每个测试性状检测到3.6667个,变幅介于1~8个。Shannon's多样性指数(H')平均值为0.6834,变幅介于0~1.9327,77.08%的性状存在3个及以上的等位变异;地方品种的等位变异数、每个性状的等位变异数及其变异幅度、多样性指数平均值等均大于现代育成品种,在外观形态上,地方品种的遗传多样性比现代育成品种更丰富。利用UPGMA法进行聚类分析发现在相似系数为0.53时,可将186个粳稻品种分为3个类(亚)群,现代育成品种单独组成一个亚群;而主坐标分析将186个粳稻品种明显的分为地方品种和现代育成品种两大区。地方品种具有丰富的多样性,与现代育成品种具有明显的形态差异,可为今后粳稻育成品种的改良提供更多的形态基础。     

ACE、CYP11B2基因多态性与慢性心力衰竭患者醛固酮脱逸的关系

目的:研究CYP11B2-344C/T(醛固酮合成酶)及ACEI/D(血管紧张素转化酶)基因多态性与慢性心力衰竭(CHF)患者实施ACEI治疗后出现醛固酮脱逸表现的关系。方法:回顾分析2008年10月至2012年10月我科收治的252例CHF患者,全部患者应用ACEI治疗3月,醛固酮在基线以上为醛固酮脱逸,依据此标准将患者分为研究组(脱逸组,n=86)与对照组(非脱逸组,n=166),依据PCR(聚合酶链反应)及RFLP(片段长度限制多态性)等方法分别检测两组CYP11B2及ACE基因型,比较两组基因型频率的分布。结果:252例患者中,共86例出现醛固酮脱逸,发生率为34.1%。全部受试患者CYP11B2基因型及ACE基因型频率与Weinberg-Hardy平衡均相符(P均0.05)。研究组ACE I/D三种基因型的组间分布与对照组相较,无统计学差异(P0.05);CYP11B2基因TT型的频率与对照组相较,呈明显统计学差异(P0.05),等位基因C/T频率的组间分布同对照组相较,亦呈明显差异(P0.05)。研究组ACEI/D的基因多态性及CYP11B2-344C/T的多态性中,基因型联合组间分布与对照组相较,无统计学差异(P0.05)。结论:ACE基因多态性与CHF患者ACEI治疗后出现醛固酮脱逸无关,CYP11B2基因T等位基因及TT基因型多态性可能是CHF患者ACEI治疗后发生醛固酮脱逸的高危因素。醛固酮脱逸时,ACE、CYP11B2基因不具有协同效果。     

Molecular Cytogenetic Identification of a New Wheat-Rye 6R Chromosome Disomic Addition Line with Powdery Mildew Resistance

Rye (Secale cereale L.) possesses many valuable genes that can be used for improving disease resistance, yield and environment adaptation of wheat (Triticum aestivum L.). However, the documented resistance stocks derived from rye is faced severe challenge due to the variation of virulent isolates in the pathogen populations. Therefore, it is necessary to develop desirable germplasm and search for novel resistance gene sources against constantly accumulated variation of the virulent isolates. In the present study, a new wheat-rye line designated as WR49-1 was produced through distant hybridization and chromosome engineering protocols between common wheat cultivar Xiaoyan 6 and rye cultivar German White. Using sequential GISH (genomic in situ hybridization), mc-FISH (multicolor fluorescence in situ hybridization), mc-GISH (multicolor GISH) and EST (expressed sequence tag)-based marker analysis, WR49-1 was proved to be a new wheat-rye 6R disomic addition line. As expected, WR49-1 showed high levels of resistance to wheat powdery mildew (Blumeria graminis f. sp. tritici, Bgt) pathogens prevalent in China at the adult growth stage and 19 of 23 Bgt isolates tested at the seedling stage. According to its reaction pattern to different Bgt isolates, WR49-1 may possess new resistance gene(s) for powdery mildew, which differed from the documented powdery mildew gene, including Pm20 on chromosome arm 6RL of rye. Additionally, WR49-1 was cytologically stable, had improved agronomic characteristics and therefore could serve as an important bridge for wheat breeding and chromosome engineering.     

The Use of Three Long Non-Coding RNAs as Potential Prognostic Indicators of Astrocytoma

Long noncoding RNAs (lncRNAs) are pervasively transcribed and play a key role in tumorigenesis. The aim of the study was to determine the lncRNA expression profile in astrocytomas and to assess its potential clinical value. We performed a three-step analysis to establish the lncRNA profile for astrocytoma: a) the lncRNA expression was examined on 3 astrocytomas as well as 3 NATs (normal adjacent tissues) using the lncRNA microarray; b) the top-hits were validated in 40 astrocytomas (WHO grade II-IV) by quantitative real time-PCR (qRT-PCR); c) the hits with significant differences were re-evaluated using qRT-PCR in 90 astrocytomas. Finally, 7 lncRNAs were found to have a significantly different expression profile in astrocytoma samples compared to the NAT samples. Unsupervised clustering analysis further revealed the potential of the 7-lncRNA profile to differentiate between tumors and NAT samples. The upregulation of ENST00000545440 and NR_002809 was associated with advanced clinical stages of astrocytoma. Using Kaplan-Meier survival analysis, we showed that the low expression of BC002811 or XLOC_010967, or the high expression of NR_002809 was significantly associated with poor patient survival. Moreover, Cox proportional hazard regression analysis revealed that this prognostic impact was independent of other clinicopathological factors. Our results indicate that the lncRNA profile may be a potential prognostic biomarker for the prediction of post-surgical outcomes.     

Impact of Phyllostachys heterocycla ‘Pubescens’ expansion on mycorrhizal associations of the adjacent forests

 毛竹(Phyllostachys heterocycla ‘Pubescens’)凭借其独特的生长特性极易扩张进入周边的常绿或针阔混交森林群落并取而代之。菌根减弱假说对毛竹林扩张导致周边林分枯亡并抑制林下幼苗更新的机制进行了解释, 即毛竹林的成功扩张是由于毛竹蔓延引起森林群落的菌根系统紊乱, 使宿主植物与菌根真菌的共生关系受到干扰, 进而影响了宿主植物的分布与更新。该研究以浙江省西天目山国家自然保护区为研究区域, 对菌根减弱假说进行了检验。通过在毛竹-针阔混交林交接区沿毛竹扩张方向设置毛竹纯林、竹-林过渡带、针阔混交林3种类型的样带, 选取在针阔混交林、竹-林过渡带同时存在的6种优势乔灌树种——杉木(Cunninghamia lanceolata)、枫香树(Liquidambar formosana)、青冈(Cyclobalanopsis glauca)、柳杉(Cryptomeria fortunei)、江浙山胡椒(Lindera chienii)、毛柄连蕊茶(Camellia fraterna), 测定这6个树种在两样带中的菌根侵染频率和强度, 检测在毛竹林扩张中周边森林群落菌根的响应, 同时对比了毛竹在毛竹纯林和竹-林过渡带菌根感染率和强度的变化, 检验该假设。实验结果表明: 1)针阔混交林和竹-林过渡带的主要树种菌根都具有较高的菌根侵染频率(> 95%), 且不同林分间林木的侵染频率无显著差异(p > 0.1); 2)在竹-林过渡带杉木和江浙山胡椒的丛枝菌根侵染强度较针阔混交林明显增加(p < 0.1); 3)毛竹的丛枝菌根侵染频率和强度远低于其他针阔树种, 且在扩张前后没有显著变化(p > 0.1)。实验结果否定菌根减弱假说。     

木瓜籽毛油精炼条件筛选及其理化指标分析

以磷脂含量为指标对木瓜〔Chaenomeles sinensis ( Thouin) Koehne〕籽毛油水化脱胶过程中脱胶剂种类、脱胶剂添加量、脱胶时间、加水量和脱胶温度进行单因素实验,并在此基础上对脱胶时间、加水量和脱胶温度进行L9(33)正交实验;以酸价为指标对碱炼脱酸过程中的碱液(NaOH溶液)浓度、碱炼温度和超碱用量进行单因素实验和L9(33)正交实验;并比较了毛油、脱胶油、脱酸油和精炼油的主要理化指标变化。单因素实验和正交实验结果表明：在木瓜籽毛油水化脱胶过程中采用不同的脱胶剂种类(包括柠檬酸、草酸和蒸馏水)、脱胶剂添加量(质量分数0.1%~0.5%)、脱胶时间(10~70 min)、加水量(质量分数1%~6%)和脱胶温度(65℃~85℃),毛油中的磷脂含量均有明显差异;而碱炼脱酸过程中采用不同的碱液浓度(质量分数6%~14%)、碱炼温度(40℃~80℃)和超碱用量(质量分数0.15%~0.40%),毛油酸价也有明显变化。总体上看,木瓜籽毛油水化脱胶的适宜条件为添加质量分数0.2%柠檬酸为脱胶剂、脱胶温度75℃、加水量为质量分数4%、脱胶时间50 min;碱炼脱酸的适宜条件为碱液浓度为质量分数12%、碱炼温度80℃、超碱用量为质量分数0.30%。理化指标的测定结果表明：与毛油相比,脱胶油、脱酸油和精炼油的碘值略升高但差异不明显、过氧化值明显升高、磷脂含量和皂化值均明显下降,而脱酸油和精炼油的酸价也明显下降。研究结果显示：经过脱胶、脱酸、水洗干燥一系列过程后获得的木瓜籽精炼油的理化指标基本符合国家食用植物油卫生标准。     

绿海龟稚龟性别鉴定及临界温度

通过组织形态学鉴定、羊水和血液中性激素测定等手段,对惠东港口海龟保护区不同恒温下孵化的绿海龟(Chelonia mydas)进行研究,寻求绿海龟稚龟性别鉴定的最佳方法。结果显示,(1)组织形态学,绿海龟性腺切片分皮层和髓质两部分,卵巢的皮层较厚、基质中有大小不一的未成熟卵细胞,髓质中空腔较少;睾丸的皮层较薄,髓质中空腔较多,呈现出显著的被基质包围的髓质索。(2)性激素测定,当雌二醇与睾酮含量之比E2∶T1.5时,孵出的稚龟为雄性(♂);当E2∶T1.5时,为雌性(♀)。(3)绿海龟性别决定的临界温度在29.4～29.5℃之间,此时孵出稚龟的性比约为1∶1。本文还讨论了各种不同性别鉴定方法的优劣和时期选择。     

华中五味子ISSR-PCR反应体系优化及引物筛选

系统研究了华中五味子ISSR PCR反应体系中的主要影响因子,确立华中五味子ISSR-PCR最适反应体系,并筛选出12条有效引物。单因子实验结果显示,华中五味子ISSR-PCR反应体系中各主要成分的适宜浓度范围为,Mg²⁺ 1.50~3.50 mmol·L^-1,dNTPs 0.10~0.35 mmol·L^-1,引物0.25~0.60 μmol·L^-1,Taq酶0.50~1.50 U。4因子3水平正交实验确立了最适反应体系,即20 μL体系中包含2.50 mmol·L^-1 Mg²⁺、0.20 mmol·L^-1 dNTPs、0.25 μmol·L^-1引物、1.50 U Taq酶、60 ng DNA模板、2.50 μL 10×PCR Buffer。本研究为华中五味子种质资源的评估及遗传多样性分析奠定基础。