长白猪甘露聚糖结合凝集素A基因的 克隆与原核表达

从长白猪肝脏细胞中提取总RNA,通过RT-PCR法扩增pMBL-A基因,与pMD18-T载体连接进行TA克隆,得到猪甘露聚糖结合凝集素A（porcine mannan-binding lectin A,pMBL-A）基因。再亚克隆到pPROEXHTb表达载体上,构建重组表达质粒。将阳性重组表达质粒转化到大肠杆菌中诱导表达重组pMBL-A蛋白,通过SDS-PAGE检测蛋白的表达情况。结果成功扩增得到包括完整开放读码框长为875bp的全长cDNA片段,并原核表达了重组蛋白,为进一步研究该蛋白的遗传特征以及为猪遗传育种方面的研究提供依据。     

克隆与原核表达

 从长白猪肝脏细胞中提取总RNA，通过RT-PCR法扩增pMBL-A基因，与pMD18-T载体连接进行TA克隆，得到猪甘露聚糖结合凝集素A（porcine mannan-binding lectin A，pMBL-A）基因。再亚克隆到pPROEXHTb表达载体上，构建重组表达质粒。将阳性重组表达质粒转化到大肠杆菌中诱导表达重组pMBL-A蛋白，通过SDS-PAGE检测蛋白的表达情况。结果成功扩增得到包括完整开放读码框长为875bp的全长cDNA片段，并原核表达了重组蛋白，为进一步研究该蛋白的遗传特征以及为猪遗传育种方面的研究提供依据。     

长白猪甘露聚糖结合凝集素-C基因启动子区的克隆与序列分析

摘要：根据GenBank上已发表的猪甘露聚糖结合凝集素C基因启动子区序列,设计一对特异性引物,通过RT-PCR法成功的从长白猪肝脏中克隆到MBL-C基因启动子区序列。扩增序列全长2030bp,与已发表的该序列相比具有3个SNPs位点,即-1636位点为T,-1614位点为A,-251位点为C,在-251位点与高水平表达的pMBL-C启动子区位点不同,从A突变为C。本研究为下一步分析pMBL-C的启动子区多态性奠定了基础。     

甘露聚糖酶协同水解甘露聚糖研究进展

甘露聚糖是软质木材中的重要多糖。不同植物来源的甘露聚糖在成分、结构和复杂性上差异很大。甘露聚糖水解为单糖需要甘露聚糖酶、甘露糖苷酶、半乳糖苷酶等多种酶的协同作用,但研究其两者或三者之间的作用较少。为了进一步探究甘露聚糖酶协同水解甘露聚糖的作用机制,本研究在概述甘露聚糖来源及结构的基础上,重点论述了甘露聚糖酶的来源、种类、作用方式及产物类型,尤其是通过多种甘露聚糖酶的协同作用增加甘露聚糖的水解效率。最后,展望了甘露聚糖酶协同作用进程与机理的研究方向,为甘露聚糖酶开发利用提供基础。     

β-甘露聚糖酶的营养功能及其在动物饲料中的应用

综述了β-甘露聚糖的抗营养作用,β-甘露聚糖酶的来源与作用方式,β-甘露聚糖酶的营养与免疫等功能,以及β-甘露聚糖酶在饲料中的应用。     

植物激素对水稻种子萌发过程中β-甘露聚糖酶活性的调控

为了研究GA3和ABA对种子萌发过程中产生的β-甘露聚糖酶活性的影响,以水稻种子作为研究材料,测定了GA3和ABA存在情况下的β-甘露聚糖酶活性,结果表明:GA3和ABA不仅对水稻种子萌发有明显的促进和抑制作用,对种子萌发过程中β-甘露聚糖酶活性也有显著的影响。50μMGA3可使β-甘露聚糖酶活性出现的时间从48h提早到吸水36h,并且显著提高酶的活性。100μMABA在强烈抑制种子正常萌发的同时也抑制了β-甘露聚糖酶活性。但是50μMGA3可恢复ABA对β-甘露聚糖酶活性的抑制作用。去胚的半粒水稻种子在水中吸涨时检测不到β-甘露聚糖酶的活性,但在水中加入50μMGA3后,可恢复去胚半粒种子产生酶活性的能力。因此,推测水稻种子萌发时β-甘露聚糖酶活性的产生可能是由胚中而来的某种物质所诱导,而这种物质很可能就是GA3。     

魔芋甘露聚糖的加工

魔芋的块茎经加工后,可制成魔芋片、魔芋豆腐、魔芋粉。在魔芋精粉中含有甘露聚糖55～60％。魔芋甘露聚糖,具有扩张毛细血管,降低血压,降低胆固醇的作用,是医药、化工原料。     

鱼类凝集素概述

鱼类凝集素成员种类繁多、功能多样,是鱼体免疫系统的重要组成部分。随着生物技术的发展,人们不断从鱼体内分离出多种具有生物活性的凝集素,对鱼类凝集素的研究逐年增多。以鱼类凝集素为研究对象,结合国内外研究成果,对鱼类凝集素的分类及功能等进行阐述,以期为今后的研究及应用提供一定的参考。     

植物β-甘露聚糖酶的研究现状

β-甘露聚糖酶（EC3．2．1．78）是一种内切水解酶，主要分解细胞壁半纤维素组分中的甘露聚糖，它在植物生长发育中的作用引起了较多的关注。本文从β-甘露聚糖酶的性质及它在种子萌发、果实成熟和花粉发育中的生理功能和相关基因克隆研究近况作一综述，以期为进一步深入研究和探讨β-甘露聚糖酶与植物生长发育的关系提供依据。     

脐橙离区β-甘露聚糖酶基因片段克隆和序列分析

β-甘露聚糖酶是一种参与细胞壁半纤维素组分甘露聚糖降解的关键酶。为探究该酶与器官脱落的关系,以纽荷尔脐橙（Citrus sinensis Osbeck）果柄离区为材料,采用同源PCR克隆策略,扩增脐橙β-甘露聚糖酶基因片段。结果表明：该序列长度274 bp,编码了一段由91个氨基酸残基组成的β-甘露聚糖酶保守区片段。同源比对表明,该序列与GenBank上其他已有的植物β-甘露聚糖酶氨基酸序列的同源性在53%~65%,推断克隆得到的片段是脐橙β-甘露聚糖酶的cDNA片段。     

长白猪甘露聚糖结合凝集素-C基因的克隆与序列分析

摘 要：从长白猪肝脏组织中提取总RNA,经RT-PCR技术扩增pMBL-C基因,与pMD18-T载体连接,获得pMBL-C基因。序列测定结果显示该基因编码区为723bp,编码241个氨基酸残基。与已发表的猪MBL-C具有99%同源性;进化树分析显示与哺乳动物的MBL-C基因,尤其是牛的亲缘关系最近,而与MBL-A基因相差较大,与禽类的最远。本研究为进一步研究猪MBL分子的遗传特征提供了依据。     

Characterisation of transgenic oilseed rape expressing pea lectin in anthers for improved resistance to pollen beetle

In order to evaluate pea lectin as a resistance factor against the pollen beetle (Meligethes aeneus), transgenic oilseed rape (Brassica napus) has been produced wherein the pea lectin gene expression is driven by a pollen-specific promoter. The aim of the present study was to identify and characterise non-segregating transgenic and non-transgenic lines to be compared in various future tests for benefits and risks associated with such transgenic crop plants. Three doubled haploid (DH) populations (1436, 1440 and 1451) expected to include lectin-producing lines and two DH populations (1443 and 1449) expected to be free of lectin were produced. All five populations originated from different transformation events in the cultivar Westar. The relative amounts of DNA from the marker gene cassette were quantified by conventional as well as real-time PCR analyses and lectin concentrations were estimated by western blot analysis. Two populations with high lectin concentrations, 1436 and 1451, contained higher amounts of the marker DNA and thus more lectin gene copies as compared with 1440 which had lower concentrations of the lectin. As expected all DH lines from 1443 and 1449 were free of lectin. Maximum pea lectin concentration obtained corresponded to 3% of total soluble protein in the anthers. There were significant differences between the populations with respect to bud and flowering stage phenology as well as seed yields, but the differences were not related to their transgenic status. All in all there were 171 lines tested for phenology and transgenic status, out of which 89 with similar phenology were subjected to tests for lectin concentration and seed yield, and 20 of these lines were retested in the next generation. Finally two lines with high lectin concentrations and one with an intermediate level along with two matching non-transgenic lines were selected for future benefit/risk experiments.     

山西省不同品种马铃薯凝集素质量分数分析

为研究山西省不同品种(品系)马铃薯凝集素的质量分数,通过对马铃薯凝集素粗提液进行大孔树脂柱层析法提纯,获得马铃薯凝集素溶液,然后采用测定吸光度与凝集活性效价的方法,评定了山西省大同市不同品种(品系)马铃薯凝集素质量分数高低。结果表明,大孔树脂柱层析法提纯马铃薯凝集素效果良好,滤液可以用于后续的分析测定。根据吸光值和凝血效果综合评定,不同品种(品系)马铃薯凝集素质量分数顺序为：05-32-7＞同薯23号＞晋薯27号＞同薯30号＞晋薯23号。     

黑木耳凝集素对RAW264.7细胞免疫调节作用的研究

为解决黑木耳凝集素对RAW246.7细胞分泌IL-6、IL-1β、TNF-α、NO释放量与免疫调节作用的问题,通过40%(NH₄)₂SO₄沉淀法对黑木耳凝集素进行分离提纯,并伴随凝血活性检测、氨基酸组分检测,得到黑木耳凝集素粗品。经过AKTA蛋白纯化系统得到黑木耳凝集素纯品。使用SDS-PAGE电泳对凝集素分子量进行分析。格里斯法检测巨噬细胞RAW264.7分泌NO的情况。ELISA检测IL-6、IL-1β、TNF-α的释放量。结果表明,黑木耳凝集素分子量约为20 kDa。通过检测发现,黑木耳凝集素对巨噬细胞RAW264.7分泌细胞因子IL-6、IL-1β、TNF-α有促进作用,且呈现剂量依赖,具有免疫调节的作用。     

Identification of low lectin mutants in soybean

Lectin is one of the known antinutritional factors that deteriorate the soybean protein quality and development of cultivars with low lectin content will help to improve nutritional quality of soybean [Glycine max (L.) Merrill]. Therefore, attempts were made to induce mutations for low lectin content in the cultivar ‘MACS 450’. Soybean cultivar ‘MACS 450’ was subjected to combination treatments of γ‐rays and ethyl methane sulphonate (EMS) with an objective to induce variability for low lectin content. The treatments of different combinations of γ‐rays and EMS were 50 Gy + 0.2% EMS, 50 Gy + 0.4% EMS, 100 Gy + 0.2% EMS and 100 Gy + 0.4% EMS. Of the 3200 treated M₁ seeds sown, 16 400 M₂ plants were raised. In M_2, 72 plants were identified for low lectin content [<40 × 10⁵ haemagglutination unit (HAU)/mg] and were carried up to M₅ generation. In M₅ generation, lectin content in ‘MACS 450’ was 39.23 to 50.0 × 10⁵ HAU/mg, and was compared with the nine true breeding lines identified having low lectin content, ranging from 2.3 × 10⁵ to 27.46 × 10⁵ HAU/mg. Three mutants were found to possess very low lectin content (ranging from 2.0 × 10⁵ to 3.0 × 10⁵ HAU/mg). Thus, the identified mutant lines with low lectin content will greatly improve soybean protein quality, thereby reducing financial burden on the soybean industry for processing soybean meal and also making it suitable for human consumption. All the mutants showed normal seed development, having soluble protein content similar or higher than that in the parent (32.0 mg/ml). This indicates that the change in lectin content does not have any negative impact on the plant growth and protein content.     

猪细环病毒k2型福建株ORF2基因克隆

为丰富福建省猪细环病毒的分子流行病学数据,本研究根据猪细环病毒1a型、1b型和k2型特异性检测引物对临床顽固性腹泻仔猪病料组织进行PCR检测,结果从1例病料中检测到猪细环病毒k2型阳性。并通过设计猪细环病毒k2型ORF2基因特异性引物对阳性病料扩增后进行克隆测序,获得猪细环病毒k2型ORF2基因完整编码区序列。分析发现所克隆的猪细环病毒k2型福建株ORF2基因全长为203 bp,编码有67个氨基酸。本实验株ORF2基因和猪细环病毒k2型代表株德国家猪分离株2p株(Gen Bank登录号AY823991)核苷酸同源性高达97.5%;和猪细环病毒1a型(Sd_TTV31株)和1b型(1p株)代表株核苷酸同源性均低于50.0%,分别为43.9%和47.3%。从遗传进化关系上看,本实验株ORF2基因和Gen Bank中猪细环病毒k2型处于同一遗传进化分支,而猪细环病毒1a型和猪细环病毒1b型均处于其他遗传分支。本研究首次在福建猪群中检测到猪细环病毒k2型感染。     

黑木耳凝集素的提取优化及体外抗肿瘤活性的研究

为了获得黑木耳凝集素的最优提取条件以及研究其对乳腺癌细胞MCF-7的抑制作用,采用单因素试验和响应面的方法,对提取时间、提取温度及料液比3个条件进行优化;采用CCK-8试剂法、形态学观察、划痕法来探究黑木耳凝集素对MCF-7的增殖、形态学变化和迁移率的影响来研究其抗肿瘤活性。结果表明：在浸提时间为8.47 h,料液比为1:51.71,(NH₄)₂SO₄浓度为50.23%时,粗提液中的凝集活性最高,可得最大理论值为5.4795 HU;黑木耳凝集素处理细胞后,细胞的存活率降低,细胞数量减少,细胞形态发生变化,划痕闭合速度减慢。且与凝集素浓度具有依赖性,浓度越高细胞活性越低,细胞数量越少,对细胞的迁移率抑制性越强。该实验获得了黑木耳凝集素的最优提取条件,以及对MCF-7细胞的抑制效果,这对凝集素的提取方法及其抗肿瘤活性的研究提供了参考。