哈茨木霉β-葡聚糖酶诱导、纯化及对黄瓜幼苗的促生防病作用

在实验室条件下分别以β-葡聚糖、麦麸和黄瓜枯萎病菌(Fusarium oxysporum)细胞壁作为唯一碳源诱导哈茨木霉菌(Trichoderma harzianum)Th-30发酵产生β-葡聚糖酶,采用硫酸铵盐析和琼脂糖凝胶色谱柱层析法对β-葡聚糖酶进行分离纯化,以津研4号黄瓜为试验材料,探究β-葡聚糖酶对黄瓜幼苗抗性生理指标、形态指标的影响及黄瓜常见土传病害的防控作用。结果表明：不同诱导条件下木霉Th-30发酵产β-葡聚糖酶活性差异显著,经β-葡聚糖诱导发酵的粗酶液酶活性最高,发酵72 h时达75.63 U·mL^-1;纯化的β-葡聚糖酶比活力为783.56 U·mg^-1,是粗酶液的45.32倍;5倍液和10倍液β-葡聚糖酶可显著提高黄瓜幼苗的根系活力、游离脯氨酸含量、苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、多酚氧化酶(PPO)活性以及根冠比和壮苗指数;β-葡聚糖酶10倍液处理对黄瓜立枯病、黄瓜枯萎病、黄瓜疫病、黄瓜猝倒病、黄瓜菌核病的防效为50.36%～80.63%。     

花椒提取物对桃褐腐病菌的抑制作用及酶活性的影响

以花椒提取物为试材,采用生长速率法测定了不同浓度的花椒提取物对桃褐腐病菌的抑制效果;并对处理后桃果实的苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、多酚氧化酶(PPO)活性进行检测。结果表明:不同浓度的花椒提取物对褐腐病菌菌丝的生长表现出较强的抑制效果,浓度为5.00mg/mL时抑制效果最好,抑制生长率达到96.71%,其EC50为0.504mg/mL;桃果实经提取物处理后,能显著诱导果实中抗病相关酶PAL、POD、PPO活性升高,并在整个试验过程中保持较高的水平。花椒提取物对桃褐腐病菌具有一定的抑制作用和诱导桃果实产生抗性的作用。     

UV-C 对葡萄黄烷醇类多酚时空积累、LAR 活性和组织定位的影响

 以酿酒葡萄‘赤霞珠’（Vitis vinifera L.‘Cabernet Sauvignon’）为试材,在果实发育过程中定期对植株进行UV-C 照射,分别采用分光光度计法、免疫组织定位等方法,对黄烷醇类多酚时空积累及其合成关键酶LAR（leucoanthocyanidin reductase,LAR）活性和定位进行初步研究。结果表明：UV-C 诱导果皮和果肉中总酚、黄烷醇类多酚、总黄烷–3–醇的积累,LAR 酶活性增强,且这一诱导作用有明显的照射剂量、器官/组织和发育阶段依赖性。UV-C 照射并不改变LAR1、LAR2 酶蛋白在葡萄果实中的分布,但诱导酶蛋白积累,特别在果皮及果肉维管束中,UV-C 照射导致LAR1、LAR2 酶蛋白信号明显增强。所有结果表明,UV-C 照射诱导果皮和果肉维管束中LAR1、LAR2 酶蛋白增加,诱导LAR 酶活性增强,最终导致总黄烷–3–醇和黄烷醇类多酚特异性积累。     

豇豆几丁质酶的诱导与纯化

用不同浓度的西瓜枯萎病菌的细胞壁碎片及孢子混合液处理‘早熟二号’豇豆幼苗 ,诱导几丁质酶。结果表明 ,以浓度为 0 .2 5mg/mL诱导 4d的效果最好。植株不同部位表现相同诱导活力的试验结果提示 ,西瓜枯萎病菌诱导液对‘早熟二号’豇豆几丁质酶的诱导是系统性的。经热变性 (6 0℃ ,30min)、硫酸铵沉淀 (6 0 %饱和度 ,4℃ )、亲和层析 (以再生的脱乙酰几丁质为层析介质 )纯化程序后 ,豇豆几丁质酶被纯化至均一程度 ,经SDS -PAGE电泳显示单一条带 ;N端氨基酸残基分析结果为EQCGS。     

短波紫外线(UV-C)结合冰温贮藏对蓝莓采后保鲜效果的影响

以蓝莓果实为试材,研究了5kJ·m~(-2) UV-C、冰温贮藏(-2±0.5)℃及其联合处理对蓝莓果实采后贮藏期间呼吸强度、失重率、腐烂率、超氧化物歧化酶(SOD)活性、抗坏血酸过氧化物酶(APX)活性和苯丙氨酸解氨酶(PAL)活性的影响。结果表明:与对照组(未处理蓝莓果实)相比,各处理组均能有效抑制蓝莓呼吸强度、失重率和腐烂率的上升,提高SOD、APX和PAL活性。其中,5kJ·m~(-2) UV-C和冰温贮藏(-2±0.5)℃联合处理对蓝莓果实的保鲜效果更显著。     

百合鳞茎苯丙氨酸解氨酶的分离纯化及酶学性质研究

经40 %～75 %硫酸铵分级沉淀和DEAE-Sepharose离子交换层析,从兰州百合（Lilium davidii var. unicolor）鳞茎中分离纯化苯丙氨酸解氨酶（PAL）,纯化倍数为13.19,酶回收率4.68%。纯化的PAL经 SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测为单一蛋白带,其亚基分子量约58.7 kD。酶学性质研究表明：百合鳞茎的PAL不耐碱,更不耐酸;随着水浴时间延长和水浴温度提高,PAL活性逐渐降低,但40 ℃水浴30 min后酶活性仍保留47%;以L-苯丙氨酸为底物,40 ℃下的Km值为4.1×10^-3 mol·L^-1;金属离子Mn²⁺、Fe²⁺、Cu²⁺、Fe³⁺促进PAL活性,1.0、1.0、2.0和1.0 mmol·L^-1分别为在供试范围内的最佳处理浓度,Ag⁺、Co²⁺、Ca²⁺抑制其活性,其中Ag+的抑制作用最强。     

紫外光对‘北陆’越橘转色果花青苷积累、关键酶活性及其基因表达的影响

以3年生‘北陆’越橘为试材,对发育过程中果实花青苷含量及合成酶活性变化进行研究,并进一步利用3种紫外光（UV-A、UV-B、UV-C）分别照射转色期越橘果实,测定了果实中花青苷等酚类物质的含量、生物合成酶活性及其基因表达,从底物、酶活性、基因转录水平阐明了不同紫外光对花青苷生物合成途径的影响。3种紫外光都明显诱导了果实花青苷的积累,特别是UV-C照射后果实花青苷含量提高了2.36倍。UV照射显著诱导果实PAL、UFGT酶活性升高,VcPAL、VcUFGT转录增强,但同时也抑制了DFR酶活性及基因表达,其中PAL、UFGT酶活性与花青苷的积累极显著正相关（r = 0.807**,r = 0.894**）,而DFR酶则相反（r =–0.854**）。结果表明,UV照射能诱导丙苯氨酸途径中响应紫外处理的一些关键基因的转录激活（如VcPAL,VcUFGT）或抑制（如VcDFR）,并影响相应酶活性的变化,促使花青苷等酚类物质的积累。     

乙烯利和脱落酸对北五味子木脂素及苯丙氨酸解氨酶活性的影响

以北五味子为试材,在转色期分别用400mg/L乙烯利和1 000mg/L脱落酸(ABA)处理果实,研究乙烯利和ABA对北五味子木脂素及苯丙氨酸解氨酶(PAL)活性的影响。结果表明:转色期至成熟处理,对照果实总木脂素和PAL活性均呈增加的变化趋势,成熟期果实总木脂素乙烯利和ABA处理比对照分别提高11.08%和15.11%,处理间无显著差异,与对照差异显著;CK成熟期果实PAL活性为20.21U·h-1·g-1,乙烯利和脱落酸对提高北五味子果实总木脂素含量和增加PAL活性具有促进作用,乙烯利和ABA处理的果实PAL活性分别为22.09、22.36U·h-1·g-1,处理间差异不显著。     

香菇中γ-谷氨酰转肽酶的分离纯化及其酶学性质研究

通过(NH4)2SO4沉淀、Phenyl Sepharose FF疏水层析分离纯化香菇中的γ-谷氨酰转肽酶(γ-Glutamyltranspeptidase,GGT),纯化后的GGT的比活力到达了19.59 U/mg.SDS-PAGE分析表明,GGT由分子量分别为28 kDa和60 kDa的两个亚基组成.酶学性质试验结果表明,GGT反应的最适温度为37 ℃,最适pH 值为7.6;金属离子Na+、K+和Ca2+对酶有激活作用,而Ag+、Cu2+、Zn2+和Fe3+则有抑制作用;试验范围内,GGT水解γ-glutamyl-p-nitroanilide的米氏动力学参数Km 值为2.601 μmol/mL,Vmax值为0.0287 μmol/min;组成的氨基酸中,Glu和Asp含量较高,Met和Cys含量较低.     

紫外线诱变青霉P-M1选育柚苷酶高产菌株研究

采用紫外线对野生型柚苷酶产生菌青霉P-M1进行诱变处理以提高柚苷酶的产量,经过初步筛选最佳诱变条件为:孢子悬液(107个/mL)稀释度为10-5,15 W紫外灯,垂直照射距离为30 cm,处理时间为5 min.通过透明圈初筛和摇瓶复筛选育出一株柚苷酶高产菌株青霉PU-15,酶活达到334.3 U/mL,比出发菌株提高了137.4%,将其连续传代10次测定遗传稳定性,结果显示稳定性良好,是一株比较理想的柚苷酶高产菌株.     

White light prevents increased catechin synthesis by ultraviolet irradiation in banana fruits

Irradiation of green banana fruit (Musa AAA, Cavendish subgroup, ‘Williams’) with an ultraviolet (UV)-C fluence of 720 Jm'2 increased ethylene evolution five-fold compared with controls during the first 24 h in either dark or light. The activity of phenylalanine ammonia-lyase (PAL) in extracts from the outer peel of irradiated fruit increased at least two-fold in either light or dark, but at 20°C it took 1-2 d for PAL activity to increase above the rate in controls. Irradiation did not affect the content of total phenolic compounds in outer peel in either dark or light until 4-6 d later. The content of chlorogenic acid was also not increased in either dark or light by UV-C. The catechin content of outer peel doubled in the dark within 24 h of irradiation and continued to increase six-fold during the first three days after irradiation. Exposure to white light (13.2 Wm 2) after irradiation almost completely prevented this increase in catechin content. These results show a close metabolic relationship between catechin and the bronze pigment previously reported in bananas irradiated with UV-C.     

水杨酸和紫外线对诱导采后葡萄果皮内白藜芦醇合成作用研究

以采收后的高妻、佳利酿、田野红3个成熟葡萄品种为试材,采用外源喷施法研究了水杨酸(Salicylicacid,SA)对葡萄果皮白藜芦醇(Resveratrol,Res)含量的影响;采用裂区试验对比研究不同品种、不同紫外线种类、同一紫外线不同辐射剂量对葡萄果皮Res含量的影响。结果表明,100mg/L的SA喷施处理可以显著提高3个葡萄品种果皮中的Res含量,诱导田野红的Res合成的效果显著高于高妻和佳利酿。除UV-B处理对高妻果皮中Res含量无显著影响外,其他UV-B和UV-C处理均能导致采后3个品种的葡萄果皮中Res含量的显著增加,且UV-C对Res诱导积累效果极显著地优于UV-B。辐射剂量在0～3.6kJ/m2范围内,3个葡萄品种果皮Res含量有随UV-B和UV-C辐射剂量的增加而增大趋势,但不同紫外线种类和辐射剂量对Res诱导积累效果因品种不同存在差异。     

贮藏温度对UV-C辐射葡萄果皮白藜芦醇及其糖苷含量的影响

 用剂量3.6 kJ·m^-2的紫外线C（Ultraviolet C,UV-C）辐射处理‘京蜜’葡萄成熟果实,25 ℃条件下避光孵育24 h后,控制温度为25 ℃和0 ℃贮藏,贮藏后0、0.5、1、2、3、7、10、14、24、34 d取样,HPLC法分析果皮白藜芦醇（Resveratrol,Res）及其糖苷含量。结果表明：UV-C辐射后25 ℃条件下孵育24 h能显著提高反式白藜芦醇（trans-Resveratrol, trans-Res）和顺式白藜芦醇苷（cis-Piceid, cis-PD）的含量,不能提高反式白藜芦醇苷（trans Piceid,trans PD）含量。贮藏温度对UV-C辐射葡萄果皮Res及其糖苷含量具有重要影响。无论是对照果实还是UV-C辐射处理果实,果皮中均未检测到顺式白藜芦醇（cis-Resveratrol, cis-Res）。25 ℃和0 ℃贮藏条件下对照果皮(trans-Res trans-PD、cis-PD含量低,变化幅度很小,而UV-C处理果皮中变化幅度很大,绝大部分时间显著高于对照。     

UV-C辐射对盆栽葡萄北丰叶片和邻近果穗果实白藜芦醇及其糖苷质量分数的影响

以1a生北丰(Vitis thunbergii×V.vinifera)葡萄盆栽结果树为试材,在果实始熟期采用紫外线C(Ultraviolet C,UV-C)辐射结合韧皮部环剥的方法,研究了UV-C辐射对葡萄叶片和邻近果穗果实白藜芦醇(Resveratrol,Res)及其糖苷质量分数的影响。结果表明,UV-C辐射诱导葡萄果实Res合成积累的敏感性低于叶片,辐射葡萄叶片对邻近果穗上果皮Res的影响远远小于UV-C直接辐射葡萄果穗,且辐射诱导葡萄果皮中Res质量分数与叶片中Res质量分数存在着一定的相关性。当叶片位于果穗下方时,叶片中反式白藜芦醇(trans-Resveratrol,trans-Res)、反式白藜芦醇苷(trans-Piceid,trans-PD)和顺式白藜芦醇苷(cis-Piceid,cis-PD)质量分数表现出环剥处理显著高于不环剥处理,然而果皮中的结果正好相反。当叶片位于果穗上方时,果皮中trans-Res、trans-PD和cis-PD质量分数表现出不环剥处理显著高于环剥处理,暗示葡萄Res可能存在韧皮部运输途径,且运输方向是双向的。此外,UV-C直接辐射葡萄果穗处理果皮trans-Res和cis-PD质量分数显著高于对照和UV-C辐射葡萄叶片处理。种子中仅检测到trans-Res,且仅环剥处理和辐射下方叶片不环剥处理显著高于对照。     

葡萄离体叶片和果实白藜芦醇及其糖苷合成对UV-C辐射诱导的响应

以‘北丰’和‘北全’葡萄离体叶片和果实为试材,研究了葡萄不同器官内白藜芦醇（Res）及其糖苷合成对紫外线C（UV-C）辐射诱导的响应,结果表明：不同器官反式白藜芦醇（trans-Res）合成积累对UV-C辐射诱导反应存在差异。叶片适宜的UV-C辐射剂量为1.8 kJ · m-2,而果皮为1.8 ~ 5.4 kJ · m-2,果实Res合成积累对UV-C辐射诱导的敏感性低于叶片。3.6 kJ · m-2 UV-C辐射果粒对种子中trans-Res含量没有显著影响,直接辐射完整种子或剖开种子切面也不能显著提高trans-Res含量。UV-C辐射处理的叶片和果皮中trans-Res含量随辐射后常温避光孵育时间的延迟表现出先升后降的趋势,而反式白藜芦醇苷（trans-PD）和顺式白藜芦醇苷（cis-PD）含量在12 ~ 48 h内迅速提高,之后维持在相对稳定状态。种子中trans-Res含量在整个孵育过程中与对照之间均不存在显著差异。     

不同时长UVA照射对黄瓜霜霉病发生和生理特征的影响

以“津优35号”黄瓜为试材,在日光温室中采用不连续照射方式对黄瓜植株进行夜间辐照处理,UVA照射光强为60μW·cm^-2,照射时间分别为0、3、4、5 h·d^-1,以不辐照为对照,以期探明不同照射时间对黄瓜霜霉病发生及植株生长的影响。结果表明:随着UVA照射时间增加,促进黄瓜生长及抗霜霉病的作用更加明显。UVA照射20 d后防效高达85.92%。随着UVA照射时间延长光合色素含量整体上均增加;UVA照射5 h·d^-1的处理,株高、茎粗、叶面积分别比CK高出36.05%,42.17%,47.01%。UVA照射5 h·d^-1处理的果实横径、单果质量、果形指数分别比CK高出94.44%、84.21%、18.60%。在5 h·d^-1的紫外光UVA辐射下,处理14 d和21 d后,过氧化物酶(POD)活性均显著高出CK 80.53%、89.12%。UVA辐射5 h·d^-1,处理21 d后,苯丙氨酸解氨酶(PAL)活性显著高于0、3、4 h分别是4.64 U·g^-1·min^-1>2.48 U·g^-1·min^-1>3.68 U·g^-1·min^-1>4.35 U·g^-1·min^-1。夜间辐照5 h·d^-1下,处理7、14、21 d时类黄酮含量与CK相比均有显著的差异分别比CK高出20.77%、67.42%、89.37%。     

底物、末端产物对离体银杏叶苯丙氨酸解氨酶活性的影响

苯丙氨酸处理浓度高于2.5mmol/L时抑制酶活性,低于2.5mmol/L时促进酶活性,诱导酶活性最强的是1.25mmol/L苯丙氨酸处理。4个浓度的反式肉桂酸处理都能抑制酶活性,100mg/L反式肉桂酸抑制酶活性作用最强。3,5-二氢肉桂酸除100mg/L微弱地增加酶活性外,其余都抑制酶活性,抑制酶活性最强的为200mg/L3,5-二氢肉桂酸处理。阿魏酸除100mg/L浓度增加酶活性外,其它浓度处理都降低酶活性,50mg/L抑制酶活性最强。4个浓度的4,5,7-三羟黄烷酮(Naringenin)都能抑制酶活性,其中,6.25mg/L抑制能力最强。     

Relationship between polymorphism of angiotensin I converting enzyme gene insertion/deletion and ACE,PAI-1 activity in patients with myocardial infarction

AIM:To explore the relationship between polymorphism of angiotensin I converting enzyme gene insertion/deletion (I/D) and ACE, PAI-1 activity in patients with myocardial infarction (MI). METHODS:Ninety-three patients with MI and eighty-seven healthy controls were tested. ACE genomic DNA was amplified using the polymerase chain reaction (PCR). Serum ACE activity was measured by colorimetry, plasma level of PAI-1 activity was determined by spectrophotometric assay. RESULTS:① The frequency of ACE DD genotype and D alleles (32.3% and 54.3%) in MI group was significantly higher than those in control group (12.6% and 37.4%, P<0.01, respectively). ② The ACE activity in serum (216.00±58.26)U/L and plasma PAI-1 activity (0.85±0.19)AU/mL in MI group were significantly higher than those in control group (170.19±48.99)U/L, (0.66±0.20)AU/mL, P<0.01, respectively. The serum ACE activity was positively correlated with plasma PAI-1 activity both in MI group and control group (r=0.7108 and r=0.7829;P<0.01, respectively). ③ In MI group, the serum ACE activity and plasma PAI-1 activity showed a significantly higher level in subjects with DD genotype (251.64±57.76)U/L, (0.96±0.16)AU/mL than those with ID (211.47±51.87)U/L, (0.82±0.18) AU/mL and Ⅱ genotypes (179.84±52.65)U/L, (0.71±0.17)AU/mL. The serum ACE activity and plasma PAI-1 activity were significantly higher in subjects with ID genotype than those with II genotype (P<0.05). In control group, the serum ACE activity and plasma PAI-1 activity showed a significantly higher level in subjects with DD genotype (195.53±54.76)U/L, (0.78±0.20)AU/mL than the subjects with Ⅱ genotype (154.98±52.74)U/L, (0.59±0.17)AU/mL (P<0.05). CONCLUSION:The increased ACE activity caused by DD polymorphism may play an important role in elevating the level of plasma PAI-1. The DD genotype of ACE is associated with high PAI-1 level. The genetic variation of ACE contributes to the balance of fibrinolytic pathway, indicating the pathogenesis mechanisms linking to the ACE I/D genotype and MI.     

The inhibitory action of rhTRAIL on mouse breast carcinoma

AIM:To explore the inhibitory action of recombinant human tumor necrosis factor-related apoptosis-inducing ligand(rhTRAIL) on mouse breast cancer.METHODS:Each mouse was inoculated 0.2 mL (1×106) D2F2 cells subcutaneously in the right lower limb and they were divided into five groups randomly.The control group was infused PBS 0.2 mL, while the low-dose, medium, high groups received purified rhTRAIL 2.5 mg/kg, 5.0 mg/kg, 10.0 mg/kg, respectively, the positive group was administered cyclophosphamide 30.0 mg/kg.Every group was operated by peritoneal injection once a day for fifteen days.The mice were weighed every day.The growth state was viewed and the size of the tumor was measured every 3 d to calculate the tumor volume and tumor suppression rate.All mice were killed after 15 d.The pathologic changes of the tumor were observed under light-microscopy and electronic microscopy.The cell cycle and apoptosis index of D2F2 cells were analyzed by flow cytometry.RESULTS: The body weight and tumor volume in low-dose, medium, high groups were lower than those in control group and the restriction effect was more significant than that in the control group (P<0.01).The body weight and tumor volume in low-dose, medium, high groups decreased with the increase in rhTRAIL concentration.The difference was significant (P<0.01).Both apoptosis and necrosis of tumor cells were observed in low-dose, medium, high groups.The area of cell apoptosis was large and most area didnt have tumor cells but only apoptotic bodise.The results of flow cytometry showed that rhTRAIL induced the apoptosis of D2F2 cells, and the apoptosis rates were 7.56 %, 21.37 %, 27.16 % respectively when rhTRAIL doses were 0.25 mg/L, 0.5 mg/L, 1.0 mg/L.Significant differences among three groups were observed (P<0.05).CONCLUSION:rhTRAIL induces apoptosis in mouse breast cancer cells and even the necrosis of tumor cells.rhTRAIL has significant effect on inhibiting the growth of D2F2 cells.