Expression Patterns of the Serine Protease Gene AlSP 3 in Apolygus lucorum (Hemiptera: Miridae) at Different Temperatures

As an important digestive enzyme gene, the serine protease gene AlSP 3 plays a key role in Apolygus lucorum for digesting conventional cotton. To understand the effect of different temperatures on the expression level of AlSP 3, we analyzed the expression patterns of AlSP 3 in A. lucorum reared at different temperatures using real-time polymerase chain reaction. The results showed that at the normal temperature of 24 ℃, the expression of AlSP 3 in A .lucorum at different nymphal stages was similar. The expressions of AlSP 3 in the female adult at the pre-mating stage and the male adult at post-mating stage were 19.71- and 7.16-fold higher than their control at the 1^st nymphal stage, respectively, which were significantly higher than at any other stages. However, expression in the 4^th to 5^th A. lucorum at the nymphal stage increased with rising temperature in the range of 21～30 ℃. At 30 ℃, the expression of AlSP 3 in the 2nd to 5^th A. lucorum was the highest. The expression levels of AlSP 3 in adult A. lucorum reared at different temperatures during the new emergence and pre-mating stages were significantly higher than that of the control reared at 24 ℃ (P < 0.01), while the expression during the post-mating stage at 18℃ was higher than that at other temperatures. When incubated at extreme temperatures (4 ℃ and 40 ℃), the expression of AlSP 3 in female and male adult A. lucorum was significantly higher than that at 24 ℃ (P < 0.01). Taken together, our results showed that the external environment temperature and the internal stage of A. lucorum or its sex all had significant effects on the expression of AlSP 3 in A. lucorum (P < 0.01), and that there was some interactions between them. Therefore, AlSP 3 is very important for the female adult A. lucorum to obtain nutrition at the early stages and temperature is an important limiting factor for the expression of AlSP 3.     

黄海黄杆菌YS-9412-130低温碱性蛋白酶的基因克隆和序列测定

黄海黄杆菌YS－9412－130具有稳定的分泌低温碱性蛋白酶的能力。将其染色体DNA用Sau3AⅠ部分酶切后，低熔点琼脂糖回收2－10Kb的DNA片段，用Klenow大片段酶半补齐，与用SalⅠ酶切且半补齐的质粒pUC19连续后，转化E.Coli.JM109，构建基因文库。且酪蛋白平板法和酶联免疫吸附（ELISA）的活性筛选方法从基因文库中筛选到一株产海洋低温碱性酶的阳性克隆（命名为pHH1）。序列测定分析表明，此重组质粒包含有长度为768bp低温碱性蛋白酶基因的完整的开放读码框架（ORF）和上游基因调控序列。此片段编码由256个氨基酸组成的酶，计算分子量为83000Dal。通过Southern杂交证实了此片段来自于黄海黄杆菌YS－9412－130基因组DNA。     

Effect of Setting Condition on the Gel-Forming Ability of Rohu

To investigate the setting condition of the gel-forming ability of rohu, optimum setting temperature for strong and weak gels of unwashed and washed rohu gel and optimum setting time for maximum proteolytic activity were investigated. Nine setting temperatures were studied for textural properties and trichloroacetic acid (TCA)-soluble peptide contents. Both unwashed gel (UW-gel) and washed gel (W-gel) showed similar optimum setting conditions for producing a strong gel that was set at 40°C for 30 min, followed by heating at 90°C for 20 min. They displayed different optimum setting conditions for weak gel. Weak gel from the degradation of UW-gel and W-gel formed at 65 and 60°C, respectively. The occurrence of protein degradation of W-gels during setting at 60°C suggested that washing did not remove the endogenous protease, and the degradation of unwashed and washed mince was due to water-soluble protease and myofibril-bound protease, respectively. Eight setting times for maximum proteolytic activity were shown by the TCA-soluble peptide contents, accompanying the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern. Both gels had similar results for setting for 120 min.     

Effects of dietary coated protease on growth performance,feed utilization,nutrient apparent digestibility,intestinal and hepatopancreas structure in juvenile Gibel carp (Carassius auratus gibelio)

This study was conducted to investigate the effects of dietary protease on growth performance, feed utilization, whole‐body proximate composition, nutrient digestibility, intestinal and hepatopancreas structure of juvenile Gibel carp, Carassius auratus gibelio (mean weight 8.08 ± 0.18 g). Six diets were prepared, including a positive control diet (dietary protein 350 g/kg, PC), one negative control diet (dietary protein 33 g/kg, NC) and four protease supplementations diets, which were 75, 150, 300 and 600 mg/kg protease NC diet. After 12 weeks of diet feeding in indoor recycle aquarium tanks, no significant difference (p > .05) was found on growth performance between fish fed diet with 75–600 mg/kg protease and the PC group. Compared with the fish fed the NC diet, the specific growth rate of fish fed 300 mg/kg protease increased significantly (p < .05), as well as protein efficiency ratios (p < .05), while feed conversion was the opposite (p < .05). The nutrient digestibility of crude protein and lipid was higher (p < .05) in fish fed 150 mg/kg protease diet than the PC diet. Whole‐body proximate composition of fish was not affected (p > .05) by the dietary treatment. Serum alkaline phosphatase and albumin were significantly affected by dietary protease (p < .05), while the content of total protein, glucose, triglyceride, total cholesterol, aspartate aminotransferase and alanine aminotransferase activities in serum was not affected (p > .05). Foregut muscular thickness was thinner (p < .05), when the fish fed diets supplementation of protease in 150 or 600 mg/kg diet than the NC diet. Protease activities in hepatopancreas and foregut were higher (p < .05), in the fish fed 150 or 300 mg/kg protease diet than the fish fed the PC diet, but those in the mid‐ and hindgut were not significantly affected (p > .05) by the dietary treatments. Based on the regression analysis of weight gain rate, the optimal dietary inclusion level of protease was 400 mg/kg in the diet for juvenile Carassius auratus gibelio.     

海水养殖废水浇灌下不同盐生经济植物生理特征及其比较

采用大田试验,研究了无施肥条件下海水浇灌(对照)、1∶1海水养殖废水(1∶1养殖废水处理)、全海水养殖废水(全养殖废水处理)处理以及施肥条件下海水浇灌(施肥处理)对海蓬子和碱蓬植株生长与生理特性的影响。结果表明:1∶1养殖废水处理的植株地上部干重(DW)显著高于对照,但全养殖废水处理植株地上部DW低于对照;施肥处理植株地上部DW明显高于所有对照和其他处理,随着生育期的延长,上述效应愈加明显,尤其体现在碱蓬植株上。与施肥处理相比,两配比养殖废水灌溉抑制了2种植物生长,降低了植株地上部氮磷(NP)含量、叶绿素含量(CHL)含量和可溶性蛋白(SP)含量,并随养殖废水配比上升而降低越显著;相反,2种植物细胞相对电导率(REC)、丙二醛(MDA)含量却随着养殖废水配比的上升而升高,碱蓬的上升比海蓬子更为明显。与对照相比,1∶1养殖废水不同程度提高了2种植物DW、CHL含量、植株地上部NP含量、内肽酶、羧肽酶、超氧化物歧化酶(SOD)及过氧化物酶(POD)活性以及SP和游离氨基酸(FAA)含量;并且,随着养殖废水配比的上升,2种植物以上各指标却不同程度地下降。综上所述,1∶1养殖废水处理可有效促进2种植物叶绿素合成,提高SOD、POD活性,降低MDA积累量,减少细胞膜受损,进而加快植株生长,增加NP吸收。2种植物相比,碱蓬对海水养殖废水浓度的生理响应较海蓬子敏感。1∶1养殖废水处理可很大程度上替代肥料的施用,从而降低环境污染。     

碱性蛋白酶酶解丰年虫卵蛋白工艺条件优化

以丰年虫卵为原料,利用碱性蛋白酶酶解制备营养价值更高的多肽,并对其水解参数进行优化.以水解度(DH)为指标,通过单因素试验研究酶解时间、酶解温度、pH、底物质量分数及加酶量等因素对碱性蛋白酶酶解丰年虫卵蛋白质过程的影响.在此基础上利用3因素(pH、酶解温度和加酶量)中心组合试验设计,得出最优酶解时间为150 min,底...     

长薄鳅幼鱼消化道指数和消化酶活性研究

测定了长薄鳅(Leptobotia elongate Bleeker)幼鱼(8.0～110.1 g)的消化道指数和不同体重组(10 g、30 g、50 g)幼鱼的胃、肝胰脏、前肠、中肠、后肠的淀粉酶、胃蛋白酶、胰蛋白酶及脂肪酶活性。结果:(1)长薄鳅幼鱼肝指数平均为(1.400±0.004)%,比肠长平均为(0.418±0.080),体重与体长呈幂函数相关,相关式为:W=0.019L2.818 1(R2=0.948,P<0.01,n=43);鱼体肥满度在30 g组中最高。(2)各部位消化酶活性均以蛋白酶>脂肪酶>淀粉酶;各体重组淀粉酶活性在肝胰脏中最高(P<0.05);蛋白酶活性在胃和中肠中较高;脂肪酶活性沿消化道从前至后升高;(3)各部位淀粉酶活性在各体重组间变化不大,胃蛋白酶、肠段胰蛋白酶和脂肪酶活性随体重增加而下降。本研究认为长薄鳅幼鱼为杂食、偏肉食性,其消化酶活性存在组织特异性,体重30 g左右是其营养需求转变或者生长发育的重要时期。     

酵母培养物和芽孢杆菌对凡纳滨对虾生长、蛋白酶活性和免疫性能的影响

研究了饲料中添加酵母培养物(益康XP)和芽孢杆菌(Bacillus)制剂对凡纳滨对虾(Litopenaeusvannamei)生长、肌肉成分、蛋白酶活性和免疫功能的影响。在基础饲料(对照组)中分别添加0.075%益康XP、0.100%益康XP、0.200%芽孢杆菌,饲养均重(5.57±0.21)g的凡纳滨对虾45 d。结果表明,添加0.075%益康XP、0.100%益康XP、0.200%芽孢杆菌分别提高凡纳滨对虾增重率17.24%、12.29%、20.18%,降低饲料系数14.28%、9.77%、12.03%,提高蛋白质效率16.49%、10.05%、12.98%(P<0.05);添加酵母培养物和芽孢杆菌对凡纳滨对虾肌肉组成成分没有影响(P>0.05);0.075%益康XP组、0.100%益康XP组、0.200%芽孢杆菌组肝胰脏蛋白酶活性较对照组分别提高25.24%、15.22%、54.92%(P<0.05);各试验组肠蛋白酶活性与对照组相比有所提高,但均未达到显著水平(P>0.05);0.100%益康XP、0.200%芽孢杆菌组凡纳滨对虾血清酚氧化酶活性、溶菌酶活性和超氧化物歧化酶活性均显著高于对照组(P<0.05);攻毒实验表明,感染溶藻弧菌后,芽孢杆菌组对虾第1、3、4天的累积死亡率均显著低于对照组(P<0.05),第4天时的免疫保护率达到27.1%,而添加益康XP对累积死亡率无显著影响(P>0.05)。综上所述,饲料中添加酵母培养物益康XP和芽孢杆菌均能改善凡纳滨对虾生长性能和消化功能,芽孢杆菌能增强凡纳滨对虾对溶藻弧菌的抗感染功能。     

草鱼鱼肉蛋白酶解物抗氧化性及功能特性研究

为全面了解水解度(DH)、蛋白酶种类对草鱼鱼肉蛋白酶解产物抗氧化性和功能特性的影响,采用木瓜蛋白酶及Alcalase 2.4L在各自最适条件下进行酶解,制备水解度为10%和20%的酶解产物,对其功能特性进行分析。结果显示:随着水解度升高酶解产物的亚铁离子螯合能力增强,但还原力和清除DPPH自由基的能力下降(P<0.05)。相同水解度下与Alcalase 2.4L酶解产物相比,木瓜蛋白酶酶解产物具有较强的清除DPPH自由基能力和还原力(P<0.05)。2种蛋白酶酶解产物的溶解性、乳化性、起泡性均在pH4时达到最低,而后随pH升高而增大。相同pH下随着水解度的升高酶解产物的溶解性增强,乳化性下降。相同pH及水解度下木瓜蛋白酶酶解产物的溶解性和起泡性小于Alcalase 2.4L酶解产物,但乳化性优于Alcalase 2.4L酶解产物。酶解产物的抗氧化性及功能特性受水解度及蛋白酶种类的影响。     

水稻纹枯菌总 DNA 提取方法的比较

DNA的浓度,尤其是DNA的纯度严重影响后续基因组酶切等结果。为寻找一种高质量的水稻纹枯菌总DNA的提取方法,采用CTAB法、CTAB改良法、SDS法、蜗牛酶法、蛋白酶K法5种方法分别提取水稻纹枯菌总DNA。通过紫外吸光度测定DNA的纯度和浓度、琼脂糖凝胶电泳、PCR扩增、限制性内切酶酶切反应及Southern杂交评价DNA的质量,最终得出蛋白酶K法是获取高质量水稻纹枯菌基因组DNA的最佳方法。