北极狐β-防御素103基因 (CBD103) 启动子活性及转录调控元件分析

本研究旨在筛选调控北极狐毛色基因CBD103的启动子活性区域及转录因子结合位点,为揭示CBD103基因调控北极狐毛色形成的分子遗传机制提供依据。克隆获得了北极狐CBD103基因5′侧翼区2 123 bp的片段,并构建了4个不同长度的启动子缺失片段表达载体,通过双荧光素酶检测系统对启动子活性进行检测。对启动子活性最高区域预测出的3个特异性蛋白1 (Sp1)转录因子结合位点分别进行点突变并构建3个突变载体,利用双荧光素酶检测系统测定其活性。结果显示,在构建的4个不同长度启动子缺失片段中1 656 (-1 604/+51)区域活性最高,在此区域构建的3个突变载体的启动子活性较野生型(片段1 656)均显著降低,说明-1 604/+51区域为北极狐CBD103基因的核心启动子区,-1 552/-1 564、-1 439/-1 454和-329/-339区域为正调控区域。文中成功获得了北极狐CBD103基因的核心启动子区域和正调控区域,这为进一步研究该基因调控北极狐被毛颜色分子遗传机制奠定了基础。     

灰葡萄孢丝裂原活化蛋白激酶编码基因bmp1和bmp3的功能

【背景】植物病原真菌丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)信号途径参与病菌有性生殖、细胞壁完整、菌丝侵染、致病力、胁迫响应等过程,灰葡萄孢MAPK信号途径参与病菌生长发育、致病力以及胁迫响应,但MAPK信号途径基因在灰葡萄孢中的功能尚未完全阐明,该信号途径对灰葡萄孢的生长发育和致病力的调控机制尚不明确。【目的】明确灰葡萄孢MAPK编码基因bmp1、bmp3在病菌生长发育、致病力以及氧化胁迫响应过程的功能,为进一步阐明MAPK信号途径调控灰葡萄孢生长发育和致病力的分子机制奠定基础。【方法】利用RNAi技术构建灰葡萄孢MAPK编码基因bmp1和bmp3的RNAi突变体,并以野生型BC22菌株为对照,对bmp1和bmp3基因的RNAi突变体的表型、致病力以及对氧化胁迫的敏感性进行分析。【结果】灰葡萄孢bmp1和bmp3基因的RNAi突变体其菌落形态、菌丝形态均与野生型BC22菌株没有明显差别;bmp1基因的RNAi突变体生长速率明显减慢,分生孢子产量明显降低;bmp3基因的RNAi突变体的生长速率与野生型BC22菌株没有明显差别,不能产生分生孢子。bmp1和bmp3基因的RNAi突变体在番茄果实的表面均不能产生明显的致病症状,而且不能穿透玻璃纸。bmp1基因的RNAi突变体在含有H_2O_2的培养基上受抑制的程度显著低于野生型,而在含甲萘醌的培养基上受抑制的程度显著高于野生型;bmp3基因的RNAi突变体在含有H_2O_2和甲萘醌的培养基受抑制的程度均显著高于野生型。【结论】灰葡萄孢bmp1基因正调控病菌生长、分生孢子形成、致病力和穿透能力,参与调控病菌对氧化胁迫的响应;灰葡萄孢bmp3基因正调控病菌分生孢子形成、致病力、穿透能力以及对氧化胁迫的响应。     

A study of genomic polymorphism and diversity in sheep breeds in northeastern Anatolia

An investigation using random amplified polymorphic DNA (RAPD) markers was performed to determine the breed-specific primers and designate the RAPD fingerprints and genetic diversities of sheep breeds (Morkaraman, Akkaraman, Tuj and Hemshin) in northeastern Anatolia. The DNA samples were isolated from a total of 91 animals from four breeds, and 50 random primers were screened. Estimation of genetic relationships between the breeds revealed two clearly distinct groups of breeds: one consisted of the Morkaraman and Akkaraman breeds, and the other consisted of the Tuj and Hemshin breeds.     

Differential expression levels of aroma-related genes during ripening of apricot (Prunus armeniaca L.)

Fruit aroma is a complex trait, particularly in terms of the number of different biosynthetic pathways involved, the complexity of the final metabolites, and their regulation. In order to understand the underlying biochemical processes involved in apricot aroma, four cDNAs (Pa-aat, EU784138; Pa-adh EU395433; Pa-pdc EU395434; and Pa-lox EU439430) encoding an alcohol acyl transferase (AAT), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), and lipoxygenase (LOX), respectively, were isolated and characterized at four stages of maturity in Prunus armeniaca L. cv. Modesto. We observed a reduction in aldehyde and alcohol production between early-harvested fruit and late-harvest fruit, concomitant with an increase in ester production. qPCR analyses showed that the expression levels of the adh gene and the lox gene stayed constant at all stages. Interestingly, aat levels showed a sharp increase in the late-harvest stages concurrent with the changes observed in ester levels. The significance of these changes in relation to aroma production in apricot is discussed.     

Bromoenol Lactone Attenuates Nicotine-Induced Breast Cancer Cell Proliferation and Migration

Objectives

Calcium independent group VIA phospholipase A₂ (iPLA₂β) and Matrix Metalloproteinase-9 (MMP-9) are upregulated in many disease states; their involvement with cancer cell migration has been a recent subject for study. Further, the molecular mechanisms mediating nicotine-induced breast cancer cell progression have not been fully investigated. This study aims to investigate whether iPLA₂β mediates nicotine-induced breast cancer cell proliferation and migration through both in-vitro and in-vivo techniques. Subsequently, the ability of Bromoenol Lactone (BEL) to attenuate the severity of nicotine-induced breast cancer was examined.

Method and Results

We found that BEL significantly attenuated both basal and nicotine-induced 4T1 breast cancer cell proliferation, via an MTT proliferation assay. Breast cancer cell migration was examined by both a scratch and transwell assay, in which, BEL was found to significantly decrease both basal and nicotine-induced migration. Additionally, nicotine-induced MMP-9 expression was found to be mediated in an iPLA₂β dependent manner. These results suggest that iPLA₂β plays a critical role in mediating both basal and nicotine-induced breast cancer cell proliferation and migration in-vitro. In an in-vivo mouse breast cancer model, BEL treatment was found to significantly reduce both basal (p<0.05) and nicotine-induced tumor growth (p<0.01). Immunohistochemical analysis showed BEL decreased nicotine-induced MMP-9, HIF-1alpha, and CD31 tumor tissue expression. Subsequently, BEL was observed to reduce nicotine-induced lung metastasis.

Conclusion

The present study indicates that nicotine-induced migration is mediated by MMP-9 production in an iPLA₂β dependent manner. Our data suggests that BEL is a possible chemotherapeutic agent as it was found to reduce both nicotine-induced breast cancer tumor growth and lung metastasis.     

Modulation by Neuropeptide FF of the interaction of Mu-opioid (MOP) receptor with G-proteins

The Neuropeptide FF (NPFF) system is known to modulate the effects of opioids in vivo and in vitro. In the present study, we have investigated the effect of NPFF agonists on the coupling of the Mu-opioid (MOP) receptor to G-proteins in a model of SH-SY5Y cells transfected with NPFF₂ receptor, in which the neuronal anti-opioid activity of NPFF was previously reproduced. Activation of G-proteins was monitored by [³⁵S]GTPγS binding assay and analysis of G-protein subunits associated with MOP receptors was performed by Western blotting after immunoprecipitation of the receptor. The results demonstrate that concentrations of NPFF agonists that produce a cellular anti-opioid effect, did not affect the ability of the opioid agonist DAMGO to activate G-proteins. However, at saturating concentration of agonist or when expression of receptor was high, opioid and NPFF agonists did not stimulate [³⁵S]GTPγS binding in an additive manner, indicating that both receptors share a common fraction of a G-protein pool. In addition, stimulation of NPFF receptors in living cells modified the G-protein environment of MOP receptor by favoring its interaction with α_s, α_i2 and β subunits. This change in G-protein coupling to MOP receptor might participate in the mechanism by which NPFF agonists reduce the inhibitory activity of opioids.     

皱大球蚧(Eulecanium kuwanai)和瘤坚大球蚧(Eulecanium gigantean)地理种群的RAPD遗传分化

采用RAPD技术测定分析了河北省皱大球蚧和瘤坚大球蚧8个地理种群的遗传结构。结果表明：5种引物在8个种群中共产生了65条RAPD标记,各地理种群之间没有产生各自的特征标记;不同种群间的遗传相似程度与其地理间距呈反比;种群内的遗传相似程度比种间高。Shannon信息指数表明,皱大球蚧的平均遗传多样性高于瘤坚大球蚧,分别为0.3456和0.3225说明物种不同,其变异程度也不同。     

外源保幼激素对异色瓢虫卵巢发育及生殖相关基因转录水平的影响

【目的】本研究旨在明确外源保幼激素(juvenile hormone, JH)对异色瓢虫Harmonia axyridis成虫卵巢发育以及生殖信号通路中关键基因转录水平的影响。【方法】以萝卜蚜Lipaphis erysimi饲喂的异色瓢虫雌成虫为空白对照组,以点滴同体积丙酮的雌成虫为溶剂对照组,对人工饲料饲喂的羽化后第2天的雌成虫点滴不同剂量(80, 120和160 ng/头)JHⅢ 1, 3, 5, 7和9 d后,解剖成虫卵巢,拍照并测量卵巢长度及其第一卵室的长度和宽度。利用qPCR分析在最适JHⅢ剂量(120 ng/头)处理后1, 5和9 d的异色瓢虫雌成虫生殖信号通路中关键基因JH受体methoprene-tolerant (Met)基因、krüppel homolog 1(Kr-h1)基因、卵黄原蛋白(vitellogenin, Vg)基因(Vg1和Vg2)和卵黄原蛋白受体(vitellogeninreceptor, VgR)基因表达水平。【结果】与溶剂对照组相比,点滴80和120 ng/头剂量JHⅢ可促进异色瓢虫成虫卵巢发育,其中120 ng/头剂量JHⅢ处理羽化后第2天雌成虫后7 d卵巢中出现大量卵黄沉积,与萝卜蚜饲喂的空白对照组卵巢发育状态一致,卵巢长度以及第一卵室的长度和宽度均显著高于溶剂对照组的;160 ng/头剂量JHⅢ处理则抑制卵巢发育。qPCR结果表明,120 ng/头剂量JHⅢ处理后5和9 d雌成虫中HaMet, HaKr-h1, HaVg1, HaVg2和HaVgR基因表达量较对照组(丙酮处理组)均增加,其中处理后5 dHaMet, HaVg1和HaVgR基因表达量分别为对照组的2.78, 13.14和8.28倍,处理后9 d分别为对照组的2.36,1.85和2.01倍,差异极显著。【结论】120 ng/头JHⅢ可促进异色瓢虫成虫卵巢发育,上调处理后5和9 d时雌成虫HaMet, HaKr-h1, HaVg和HaVgR基因表达量;推测这些关键基因在保幼激素调控异色瓢虫卵巢发育及卵黄形成过程发挥重要作用。     

滨海快速城市化地区土地生态安全评价与预警——以曹妃甸新区为例

对滨海快速城市化地区进行土地生态安全评价和预警是保障土地可持续利用和有效维护土地生态安全的重要内容.基于“压力-状态-响应”(P-S-R)模型,构建了滨海快速城市化地区土地生态安全评价指标体系.借助遥感数据和GIS方法,以1 km×1 km网格为评价单元并通过空间插值,获取了基于像元大小30 m×30 m的曹妃甸新区2005和2013年土地生态安全评价结果.并采用分类树方法对未来曹妃甸新区土地生态安全(LES)进行预警,共分为4种预警类型：安全但有退化趋势预警、亚安全且缓慢退化预警、亚安全且快速退化预警、不安全预警.结果表明：曹妃甸新区2005年土地生态安全平均值为0.55,处于中等安全水平,而2013年为0.52,处于中下安全水平,区域土地生态安全呈下降趋势.曹妃甸新区不安全预警及亚安全且快速退化预警区域主要分布在城市建设用地快速扩展区域,说明城市建设用地的快速扩张是导致区域土地生态安全恶化的最主要因素.工业区、十里海和南堡盐场3个乡镇处于高度危险预警状态,其不安全预警、亚安全且快速退化预警及亚安全且缓慢退化预警3者之和分别占其总面积的58.3%、98.9%和81.2%,未来应加强这些区域的土地生态管理调控.研究结果可为曹妃甸新区土地规划和生态保护提供一定参考.