芸豆凝集素受体在小鼠生殖细胞上的分布及作用研究

[目的]探讨芸豆凝集素（PHA）受体在小鼠精子和卵母细胞上的分布及作用。[方法]应用异硫氰酸荧光素（FITC）标记的芸豆凝集素作为分子探针,确定其PHA受体在小鼠精子和卵母细胞上的分布。通过不同浓度PHA处理精子和卵母细胞,研究PHA预处理对精子获能和顶体反应,以及对卵母细胞FITC—PHA染色的影响。l结果]PHA受体主要表达于小鼠精子整个尾部区域和顶体反应后的头部质膜,2mg／mlPHA预处理有抑制顶体反应的作用;卵母细胞透明带和细胞膜都有PHA受体的存在。不同浓度PHA处理卵母细胞对其透明带阿FTIC—PHA染色影响不大,但对其细胞膜染色有较大影响,且不同浓度之间差异明显。[结论]芸豆凝集素受体是小鼠精子和卵母细胞受体的重要组成部分,且在精卵质膜融合过程中发挥重要作用。     

Actions of quinolizidine alkaloids on Periplaneta americana nicotinic acetylcholine receptors

BACKGROUND: Botanical insecticides do not play a major role as crop protectants, but they are beneficial in some applications. The authors investigated the actions of naturally occurring alkaloids on insect nicotinic acetylcholine (ACh) receptors (nAChRs) by evaluating their abilities to inhibit specific binding of [³H]imidacloprid (IMI) to nerve‐cord membranes from Periplaneta americana L. Two alkaloids were also tested for their actions on nAChRs expressed by cockroach neurons using patch‐clamp electrophysiology. RESULTS: Four natural quinolizidine alkaloids (matrine, sophocarpine, cytisine and aloperine) exhibited more than 50% inhibition of [³H]IMI binding at 10 µM , although other compounds were found to have no or low inhibitory activity. The rank order of potency based on concentration–inhibition curves was cytisine > sophocarpine ≥ aloperine ≥ matrine. Patch‐clamp analysis indicated that sophocarpine and aloperine were not agonists of nAChRs expressed in P. americana neurons, yet, at 10 µM , aloperine, but not sophocarpine, suppressed ACh‐induced inward currents significantly. CONCLUSION: Three of the four natural alkaloids tested possess structural moieties that are necessary for interaction with P. americana nAChRs. Aloperine, which possesses a unique structure and showed a distinctive dose–response curve, was found to act as an antagonist. Appropriate modifications of these alkaloids might result in novel insecticidal nAChR ligands. Copyright © 2008 Society of Chemical Industry     

Production of Transgenic Anliucheng Sweet Orange (Citrus sinensis Osbeck) with Xa21 Gene for Potential Canker Resistance

Citrus canker, an epidemic quarantine disease caused by Xanthomonas axonopodis pv. citri, has brought a great damage in citrus production worldwide. Herein, a rice PRR (pattern recognition receptor) gene Xa21 together with GUS reporter gene and hygromycin phosphotransferase gene (HPT) was introduced into Anliucheng sweet orange (Citrus sinensis Osbeck) via Agrobacterium-mediated transformation of embryogenic callus. The transgenic calluses were screened on MT basal medium containing hygromycin (HYG) and detected by histochemical GUS staining. The transgenic plantlets were recovered through somatic embryogenesis pathway. The regenerated plantlets were accustomed to and maintained in the greenhouse. The transgene integration of recovered plantlets was identified by PCR and Southern blot hybridization. It showed that all the transgenic plantlets tested had undergone single copy integration, the expression of Xa21 in eight different transgenic lines detected by qRT-PCR can be divided into three grades, high for T5 and T6, middle for T4 and low for the rest. The tolerance to citrus canker disease of the three recovered transgenic lines T2, T4 and T6 was assessed by in vitro pin-puncture inoculation. The results showed that all the three transgenic lines conferred improved resistance to citrus canker bacterium infection and the T4 transgenic line displayed the highest resistance. The mechanism and feasibility of rice Xa21 in triggering innate immunity in citrus was briefly discussed.     

兰州熊蜂气味受体家族鉴定及分析

【目的】了解兰州熊蜂(Bombus lantschouensis)基因组中气味受体基因(odorant receptors,Ors)情况,为进一步分析气味受体功能提供信息,从而为研究该基因家族在熊蜂觅食、交尾、通讯等行为中的重要功能打下基础。【方法】提取兰州熊蜂胸部基因组DNA,进行Illumina高通量测序,对测序所得原始序列进行质控并拼接获得基因组序列,然后使用本地blast 2.2.28+对构建兰州熊蜂基因组本地数据库,使用已知的地熊蜂(Bombus terrestris)和意大利蜜蜂(Apis mellifera ligustica)的气味受体蛋白序列作为种子序列搜索数据库获得兰州熊蜂的气味受体序列;使用EMBOSS1.5对气味受体蛋白序列进行基本理化分析,利用GSDS2.0对家族序列的内含子与外显子位置进行分析,然后使用MEME 4.11.2对氨基酸序列进行保守基序分析,最后利用Clusta W 2.1、Trim Al 1.2、Phy ML3.0对兰州熊蜂、地熊蜂和意大利蜜蜂气味受体家族序列进行系统进化分析。【结果】共获得165个兰州熊蜂气味受体基因,包括1个非典型气味受体(olfactory receptor co-receptor,Orco)、5个假基因和159个气味受体。基因结构分析发现,气味受体家族外显子数目从4个到9个不等。其中Or 47—57的序列中外显子数量最少,为4个;Or 128—161中外显子的数量最多,为9个。根据基因结构的不同,将气味受体分为10大类型,每个类型中的序列都有相似的外显子长度与数量。Or 1—38、Or 69—85、Or 128—164这3大类所包含成员数较多(分别为38、17、37),其他7类包含成员个数都约10个,且每个类型中序列在染色体上都呈串联排布,其中Or 1—38中都包含一个较长的第一外显子。保守基序分析发现,在检索的10个保守基序中,除基序5为未知基序外,其他9个基序均包含在其保守结构域(7tm_6 domain)中。Or1—38与Or 39—46多数成员包含全部10个保守基序,基序2、3、4、9广泛存在于该家族成员序列中,这4个基序可能为该家族关键的功能区域。系统发育分析将Ors分为5个亚家族(I—V),其中亚家族II包含2种基因结构类型序列(Bl Or 97—100与Bl Or 69—85),亚家族V包含4种(Bl Or 1—46、47—57、86—95和101—107)。Bl Or 150—155与Am Or 122—139分别聚为两个分支,Bl Or 47—57与Am Or 63—65也发现类似的聚类,这表明在进化中,蜜蜂与熊蜂的Ors出现了特异性的扩张与缺失。在进化树中发现Or 115较早与其他成员分离,位于树的基部,推测该序列可能更接近气味受体家族的祖先序列。【结论】探明了兰州熊蜂的Or基因数量、基因结构及其进化关系;该基因家族在进化过程中部分成员保守基序丢失,这些结果为进一步了解Or基因功能打下了基础。     

产肠毒素大肠杆菌K88诱导仔猪炎症反应的分子机制

为探讨产肠毒素大肠杆菌（ETEC） K88感染仔猪发生炎症反应的分子机制,试验用ETEC K88灌服断奶仔猪,ELISA法检测攻毒后仔猪血清中白细胞介素8（IL-8）含量,实时荧光定量PCR方法检测淋巴结中Toll样受体2（TLR2）、Toll样受体4（TLR4）及其信号通路相关基因（髓样分化因子88（MyD88）、Toll相互作用蛋白（Tollip）、B细胞淋巴瘤因子3（Bcl3））的mRNA相对表达水平。结果发现,仔猪攻毒ETEC K88后6和24 h血清IL-8含量和淋巴结TLR2/4的表达水平均极显著或显著高于对照组（P<0.01;P<0.05）,且感染后24 h显著低于感染后6 h（P<0.05）;仔猪感染ETEC K88后24 h淋巴结中MyD88、Tollip和Bcl3的表达水平均极显著高于对照组（P<0.01）,但是感染后6 h时与对照组相比均无显著差异（P>0.05）。综上所述,ETEC K88感染仔猪可能是通过TLR2/4-MyD88信号通路产生炎症因子IL-8,促使仔猪出现炎症反应,且该炎症反应可能受Tollip和Bcl3蛋白的调控而被减弱。     

1—2月龄荷斯坦奶牛不同组织中FMDV受体亚基αv、β3、β6 mRNA转录水平的相对定量研究

【目的】建立检测牛口蹄疫病毒(FMDV)受体亚基αv、β3、β6 mRNA相对转录水平的实时定量PCR方法,分析受体αvβ3和αvβ6在不同器官组织中的转录水平。【方法】在对牛FMDV受体基因克隆和测序的基础上,设计实时定量PCR引物,以GAPDH为内参基因,采用△△Ct相对定量PCR方法检测分析FMDV受体亚基αv、β3、β6 mRNA在1—2月龄荷斯坦奶牛体内不同器官组织中的转录表达谱。【结果】αvβ3在荷斯坦奶牛24种组织中均有不同程度的转录表达;αvβ6在甲状腺、硬腭、鼻内皮、喉头、肺、食管、肾、后蹄冠状带等组织表达量较高,在唇、舌皮、软腭、气管、心脏、瘤胃、直肠、前蹄冠状带等组织转录表达量适中,在唾液腺、下颌淋巴结、肝、脾、肌肉等组织未检测到表达;αvβ6在牛体内的表达分布与FMDV组织嗜性基本一致,αvβ6受体可能是决定FMDV组织嗜性的主要功能受体,αvβ3的组织分布似乎与FMDV组织嗜性无关,但不能排除αvβ3在病毒感染过程中的作用。【结论】建立了检测FMDV受体亚基mRNA在不同器官组织中表达水平的相对实时定量PCR方法。     

Effect of Estradiol-17β on protein synthesis and degradation rates in fused bovine satellite cell cultures

Although androgenic and estrogenic steroids are widely used to enhance muscle growth and increase feed efficiency in feedlot cattle, their mechanism of action is not well understood. Further, in vivo studies indicate that estradiol (E2) affects muscle protein synthesis and/or degradation, but in vitro results are inconsistent. We have examined the effects of E2 treatment on protein synthesis and degradation rates in fused bovine satellite cell (BSC) cultures. Additionally, to learn more about the mechanisms involved in E2-enhanced muscle growth, we have examined the effects of compounds that interfere with binding of E2 or insulin-like growth factor (IGF)-1 to their respective receptors on E2-induced alterations in protein synthesis and degradation rates in BSC cultures. Treatment of fused BSC cultures with E2 results in a concentration-dependent increase (P < 0.05) in protein synthesis rate and a decrease (P < 0.05) in protein degradation rate. The pure estrogen antagonist ICI 182 780 suppresses (P < 0.05) E2-induced alterations in protein synthesis and degradation in fused BSC cultures. The G-protein coupled receptor (GPR)-30 agonist G1 does not affect either synthesis or degradation rate, which establishes that GPR30 does not play a role in E2-induced alterations in protein synthesis or degradation. JB1, a competitive inhibitor of IGF-1 binding to the Type 1 insulin-like growth factor receptor (IGFR-1), suppresses (P < 0.05) E2-induced alterations in protein synthesis and degradation. In summary, our data show that E2 treatment directly alters both protein synthesis and degradation rates in fused BSC cultures via mechanisms involving both the classical estrogen receptor (ER) and IGFR-1.