小麦种子萌发过程中类PvLEA—18的表达

利用PvLEA-18蛋白抗体,分析小麦种子在萌发过程中类PvLEA-18表达情况。Western blot表明在小麦干种胚中未检测到类PvLEA-18蛋白,而在种子萌发不同时期（12h,24h,36h)的胚和盾片组织中检测到类PvLEA-18蛋白的表达。其分子量分别约为81kD和70kD。种子萌发24h后,81kD蛋白消失,70kD的类PvLEA-18表达量也降低。     

苦瓜果实发育过程中的主要营养成分变化及种子生活力演进

为明确苦瓜的果实和种子的适宜采收时期,采用"翠中翠"苦瓜为研究对象,对苦瓜果实发育过程中的可溶性蛋白、总可溶性糖、维生素C的含量及种子生活力的变化进行动态监测。结果显示:(1)花后第14天至第26天,随着果实的迅速膨大,可溶性蛋白、总可溶性糖及维生素C含量也迅速增长;(2)种子干物质迅速积累,含水量迅速下降,种子生活力迅速提高,至花后第22天至第26天,种子生活力基本稳定。     

聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素

以苦瓜籽为材料,研究了聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素。等电点沉淀试验表明,柠檬酸、盐酸分别调节苦瓜种仁粗提液pH至6.0、4.0时,各有14.62%和32.49%的苦瓜种仁蛋白被沉淀。醋酸的等电点沉淀作用呈现阶段性特点,pH 6.0和4.0时分别有26.17%和38.72%的苦瓜种仁蛋白被沉淀。醋酸、盐酸和柠檬酸处理的1mL苦瓜种仁粗提液（pH 4.0）,1%PAA选择性沉淀碱性蛋白（等电点pI为8.65～9.30）的最佳用量分别为100μL、120μL和100μL。醋酸调节苦瓜种仁粗提液pH分别至5.0、4.0和3.0,等电点沉淀后的上清液用PAA沉淀碱性蛋白,当PAA（1%）用量为160μL/mL提取液时,pH5.0和3.0样液分别有33.77%和43.56%蛋白质被沉淀;当PAA用量为120μL/mL提取液时,pH 4.0样液中30.83%蛋白质被沉淀。PAA-蛋白质复合物溶解于碱性溶液（pH＞9.0）,当溶液NaCl浓度为3.0%时,溶液蛋白质浓度最高。PAA选择性沉淀的苦瓜种仁碱性蛋白经Sephadex G-75柱层析分离,分别在175min和300min出现主峰Ⅰ和Ⅱ。SDS-PAGE和IEF分析表明主峰Ⅰ的分子量约为30kD,pI值约为9.5,主峰Ⅱ的分子量约为10kD,pI值约为9.3。     

聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素

以苦瓜籽为材料,研究了聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素。等电点沉淀试验表明,柠檬酸、盐酸分别调节苦瓜种仁粗提液pH至6.0、4.0时,各有14.62%和32.49%的苦瓜种仁蛋白被沉淀。醋酸的等电点沉淀作用呈现阶段性特点,pH6.0和4.0时分别有26.17%和38.72%的苦瓜种仁蛋白被沉淀。醋酸、盐酸和柠檬酸处理的1mL苦瓜种仁粗提液(pH4.0),1%PAA选择性沉淀碱性蛋白(等电点pI为8.65~9.30)的最佳用量分别为100μL、120μL和100μL。醋酸调节苦瓜种仁粗提液pH分别至5.0、4.0和3.0,等电点沉淀后的上清液用PAA沉淀碱性蛋白,当PAA(1%)用量为160μL/mL提取液时,pH5.0和3.0样液分别有33.77%和43.56%蛋白质被沉淀;当PAA用量为120μL/mL提取液时,pH4.0样液中30.83%蛋白质被沉淀。PAA-蛋白质复合物溶解于碱性溶液(pH>9.0),当溶液NaCl浓度为3.0%时,溶液蛋白质浓度最高。PAA选择性沉淀的苦瓜种仁碱性蛋白经SephadexG-75柱层析分离,分别在175min和300min出现主峰Ⅰ和Ⅱ。SDS-PAGE和IEF分析表明主峰Ⅰ的分子量约为30kD,pI值约为9.5,主峰Ⅱ的分子量约为10kD,pI值约为9.3。     

α-苦瓜素基因的克隆和原核表达

α-苦瓜素(Alpha-momorcharin)是一种多功能核糖体失活蛋白,具有抗肿瘤、抗HIV-1和抗菌活性,并同时具有免疫抑制活性.利用PCR(polymerase chain reaction)技术从苦瓜基因组中扩增出了成熟的α-苦瓜素蛋白基因,并亚克隆到表达载体pET28a( )上,然后分别在BL21(DE3) 和RosettaTM(DE3)pLysS中进行表达.结果表明只在RosettaTM(DE3)pLysS中,重组α-苦瓜素基因能成功地表达出33 kD大小的重组蛋白,而且在18℃和22℃的温度下,经IPTG诱导后,能成功地表达出大量可溶性重组蛋白.利用重组蛋白的N末端带有的6×His 标签,通过Ni-NTA柱纯化获得了α-苦瓜素重组蛋白.Western杂交实验表明,纯化后的重组蛋白可与鼠抗His-Tag单克隆抗体发生特异性反应.α-苦瓜素重组蛋白的获得为进一步系统研究和改造其功能与活性奠定了基础.     

耐盐突变体小麦后代耐盐稳定性分析研究

以卫星搭载小麦种子为原始材料,利用其幼穗、幼胚诱导的愈伤组织进行耐盐突变体的筛选,对耐盐愈伤组织再生植株后代进行耐盐稳定性生理生化特性分析。结果表明：(1)耐盐系后代在土壤高盐浓度条件下,游离脯氨酸含量稳定增加,且高于对照系;(2)耐盐系再生植株后代保持较高的K^ ／Na^ 比;(3)与对照相比,种子醇溶蛋白电泳带谱中的b2,b3,b5,b7带为耐盐系所特有,b8带消失;(4)耐盐系再生植株后代可溶蛋白电泳带为26条,而对照系为23条蛋白带。其中98kD、75kD、52kD、49kD和32kD为耐盐系的特有蛋白带。而38kD和35kD蛋白带为对照系所特有。     

苦瓜的核糖体失活蛋白

核糖体失活蛋白是一类专一修饰核糖体的大亚基rRNA从而抑制蛋白质生物合成的蛋白毒素,可分为Ⅰ-型和Ⅱ-型两种类型。苦瓜中含有多种Ⅰ-型核糖体失活蛋白,如α-苦瓜素,β-苦瓜素和MAP30等,这些蛋白成分具有抗肿瘤、抗病毒和抗艾滋病等功能,因而近年来引起人们广泛的关注。对苦瓜核糖体失活蛋白的研究进展和应用前景进行了综述。     

花生种子发育和萌发过程中贮藏蛋白的合成和降解

以花生品种汕油5 2 3种子为材料,分离纯化花生球蛋白的41 kD和38.5 kD两种主要亚基及伴花生球蛋白的6 0.5 KD亚基并制备抗体.We stern blot分析表明,3种亚基在花生胚组织分化期的胚轴和子叶中就开始合成,其中60.5 kD亚基是最先在胚轴和子叶中大量合成和积累的贮藏蛋白,41 kD和38.5 kD亚基在随后的发育中积累量不断增加;种子萌发时这3种亚基的降解进程不一样,胚轴和子叶中41 kD和38.5kD亚基的降解均先于60.5 kD亚基.     

人工老化处理的卷心菜种子的热激蛋白合成

高活力卷心菜种子蛋白质合成速率比中等活力和低活力种子高很多。热激处理(42℃)下,蛋白质合成显著下降,但高活力种子的蛋白质合成能力仍然显著高于中等和低活力种子。高活力和中等活力种子主要合成分子量为70 kD和一些小分子量的热激蛋白。在低活力种子中检测不到热激蛋白的合成。4种热激蛋白（1种HSP90和3种HSP70）的Western blot检测结果表明,只有1种热激蛋白（HSP70）与种子活力有关。     

脱水敏感的黄皮种子在发育中的可溶性蛋白变化

黄皮种子的子叶与胚轴,在发育前期蛋白质合成速率均高于后期。在发育过程中子叶的可溶性蛋白含量无明显变化,但在后期能新合成少数低分子量的热不稳定蛋白,可能是引起种子萌发的水解酶类。胚轴中可溶性蛋白单位干重含量高于子叶,而其成分不随发育而变化。ABA可促进发育后期黄皮种子胚轴中20kD蛋白的合成,但不能改变种子的脱水敏感性。     

In vitro refolded napin-like protein of Momordica charantia expressed in Escherichia coli displays properties of native napin

Napins belong to the family of 2S albumin seed storage proteins and are shown to possess antifungal activity. Napins, in general, consist of two subunits (derived from single precursor) linked by disulphide bridges. Usually, reducing environment of the E. coli cytosol is not conducive for proper folding of heterodimeric proteins containing disulphide bridges. Present investigation reports for the first time expression of napin-like protein of Momordica charantia (rMcnapin) in E. coli and its in vitro refolding to produce biologically active protein. Full-length cDNA encoding napin-like protein (2S albumin) was isolated from M. charantia seeds by immunoscreening a cDNA expression library. The cDNA consisted of an open reading frame encoding a protein of 140 amino acid residues. The 36 amino acids at the N-terminus represent the signal and propeptide. The region encoding small and large chains of the M. charantia napin is separated by a linker of 8 amino acid residues. The region encoding napin (along with the linker) was PCR amplified, cloned into pQE-30 expression vector and expressed in E. coli. rMcnapin expressed as inclusion bodies was solubilized and purified by Ni2+-NTA affinity chromatography. The denatured and reduced rMcnapin was refolded by rapid dilution in an alkaline buffer containing glycerol and redox couple (GSH and GSSG). Refolded His-rMcnapin displayed similar spectroscopic properties as that of mature napin-like protein of M. charantia with 48.7% alpha-helical content. In addition, it also exhibited antifungal activity against T. hamatum with IC50 of 3 microg/ml. Refolded His-rMcnapin exhibited approximately 90% antifungal activity when compared with that of mature napin-like protein of M. charantia. Thus, a heterologous expression system and in vitro refolding conditions to obtain biologically active napin-like protein of M. charantia were established.     

Isolation of a ribosome-inactivating and abortifacient protein from seeds of Luffa acutangula

A glycoprotein with a molecular weight of 28,000 as estimated by SDS-polyacrylamide gel electrophoresis was isolated from seeds of Luffa acutangula using a procedure that involved acetone precipitation, ion exchange chromatography on CM Sepharose CL-6B and gel filtration on Sephadex G-50. In immunodiffusion studies it was found to be immunologically distinct from abortifacient proteins isolated from other members of the Cucurbitaceae family including Momordica charantia, Momordica cochinchinensis, Trichosanthes kirilowii and Trichosanthes cucumeroides. There were some differences in amino acid composition among the proteins although there was a gross similarity. The protein from L. acutangula was capable of inducing mid-term abortion in mice and inhibiting protein synthesis in a cell-free system.     

苦瓜籽蛋白的PEG/（NH4）2SO4双水相分离及抑菌作用的研究

优化试验条件影响因素,建立PEG/(NH4)2SO4双水相系统,确定出分离苦瓜籽蛋白的最适PEG6000和(NH4)2SO4质量分数分别是22%、25%,分配系数为0.089,回收率为96%。双水相分离的苦瓜籽蛋白经SDS-PAGE凝胶电泳显示,主要蛋白质是50 KD4、3 KD和10 KD亚基结合形成的多聚体12S(327 KD)球蛋白,还原条件下出现诸多小于35 kD的蛋白组分,表明蛋白质亚基是通过二硫键结合。抑菌试验显示苦瓜籽蛋白对受试细菌菌和真菌产生明显的抑菌活性,其MIC远低于苯甲酸钠,而且在不同盐离子浓度、温度、pH及紫外线等因素处理时,均具有一定的稳定性。此外试验还表明苦瓜籽蛋白也具有较强的清除羟自由基能力和总抗氧化能力。     

Trichosanthin, alpha-momorcharin and beta-momorcharin: identity of abortifacient and ribosome-inactivating proteins

The abortifacient proteins trichosanthin, alpha-momorcharin and beta-momorcharin at nM concentrations inhibit cell-free protein synthesis. The momorcharins and the ribosome-inactivating proteins isolated from Momordica charantia seeds cross-react with the respective antisera. The ribosome-inactivating proteins saporins, pokeweed antiviral protein (PAP) and, to a lesser extent, gelonin have abortifacient activity on pregnant mice.     

光照对苦瓜形态可塑性及生物量配置的影响

在人为遮阴条件下,对苦瓜的生长动态、形态特征以及生物量分配进行了研究。结果表明,不同遮阴处理下苦瓜植株的生长有较大的差异,弱光照不利于苦瓜构件数量的增加和生物量的积累;植株在弱光下形成较少的分枝,较薄的叶片以及较细长的主茎和叶柄,表现出较强光生长环境下更强的形态可塑性;植株在生长早期较生长晚期有较大的形态可塑性;生物量对叶片和主茎的分配随光照的减弱而增大,分枝的生物量分配随光照的减弱而降低,光照对分枝茎的生物量分配影响不大;在光资源充足的情况下,外界支持物的缺乏不会对苦瓜的生长造成太大的影响。     

Cytoxicity of erythrocyte ghosts loaded with ribosome-inactivating proteins following fusion with CHO cells

The ribosome-inactivating proteins gelonin, Momordica charantia inhibitor, pokeweed antiviral protein, and one from Saponaria officinalis were enclosed in human erythrocyte ghosts. The proteins once trapped in ghosts and fused with CHO cells inhibited colony formation at concentrations of approximately 1 ng/ml (3 X 10(-11) M), whereas the free proteins only had an effect at concentrations of greater than 1 microgram/ml.     

Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina

Plant seeds, a rich source of proteins, are considered important for their application as functional ingredients in a food system. A novel ribosome-inactivating protein (RIP), balsamin was purified from the seeds of Balsam apple, Momordica balsamina. Balsamin was purified by ion exchange chromatography on CM Sepharose and gel filtration on superdex-75. It has a molecular weight of 28 kDa as shown by SDS-PAGE analysis. Balsamin inhibits protein synthesis in a rabbit reticulocyte lysate-based cell free translation assay with an IC(50) of 90.6 ng ml(-1). It has RNA N-glycosidase activity and releases a 400-base long fragment termed the Endo fragment from 28S rRNA in the same manner as does saporin-6 from Saponaria officinalis. The N-terminal sequence analysis of the first 12 amino acids of balsamin revealed that it shares 83% similarity with type I RIP α-MMC from Momordica charantia and 50% similarity with β-MMC (from Momordica charantia), bryodin I (from Bryonia dioica) and luffin a (from Luffa cylindrica). Balsamin was further characterized by mass spectrometry. CD spectroscopic studies indicate that secondary structure of balsamin contains helix (23.5%), β-strand (24.6%), turn (20%) and random coil (31.9%). Thus RIPs activity expressed in vegetables like Momordica sp. advocates its usage in diet.     

Binding Energy calculation of GSK-3 protein of Human against some anti-diabetic compounds of Momordica charantia linn (Bitter melon)

Diabetes is one of the major life threatening diseases worldwide. It creates major health problems in urban India. Glycogen Synthase Kinase-3 (GSK-3) protein of human is known for phosphorylating and inactivating glycogen synthase which also acts as a negative regulator in the hormonal control of glucose homeostasis. In traditional medicine, Momordica charantia is used as antidiabetic plant because of its hypoglycemic effect. Hence to block the active site of the GSK-3 protein three anti-diabetic compounds namely, charantin, momordenol & momordicilin were taken from Momordica charantia for docking study and calculation of binding energy. The aim of present investigation is to find the binding energy of three major insulin-like active compounds against glycogen synthase kinase-3 (GSK-3), one of the key proteins involved in carbohydrate metabolism, with the help of molecular docking using ExomeTM Horizon suite. The study recorded minimum binding energy by momordicilin in comparison to the others.     

Inhibition of increases in blood glucose and serum neutral fat by Momordica charantia saponin fraction

Focusing on a functional component of Momordica charantia, saponin, we investigated its effects on serum glucose and neutral fat levels. Saponin was extracted as a butanol-soluble fraction (saponin fraction) from hot blast-dried Momordica charantia powder. The disaccharidase-inhibitory activity and the pancreatic lipase-inhibitory activity of the saponin fraction were measured, and in vivo sugar- and lipid-loading tests were performed. The saponin fraction inhibited disaccharidase activity and elevation of the blood glucose level after sucrose loading. The fraction also markedly inhibited pancreatic lipase activity and elevation of the serum neutral fat level after corn oil loading. Based on these findings, the main active component related to the anti-diabetic effect of Momordica charantia is present in the butanol fraction, and it may be saponin. The blood glucose and serum neutral fat-lowering effects of Momordica charantia were closely associated with its inhibitory activity against disaccharidase and pancreatic lipase.     

苦瓜MAP30基因的毕赤酵母表达载体构建及重组菌株的筛选

目的:从苦瓜中克隆MAP30全长基因,并将该基因连接至表达载体pPIC9中,建立酵母菌落PCR筛选方法。方法采用改良SDS法从苦瓜表皮中提取基因组DNA,设计特异性的引物,通过PCR技术扩增出全长861bp的MAP30基因。该基因经XhoⅠ和EcoRⅠ双酶切,连接至毕赤酵母表达载体pPIC9中。重组载体转化GS115菌株,运用菌落PCR鉴定重组菌株。结果:基因测序表明,该基因已成功插入酵母表达载体pPIC9α-factor分泌信号下游,同源性分析表明该基因与GeneBank(AF284811)的核苷酸同源性达99.9%,氨基酸同源性达100%。菌落PCR显示外源基因已整合入酵母GS115菌株中。结论:成功地克隆了MAP30全长基因,并构建了含MAP30基因的重组毕赤酵母表达载体,并获得了整合菌株,为下一步研究奠定了基础。