基于EMA-qPCR的茄科青枯菌活体检测技术的建立

【目的】利用特异性核酸染料叠氮溴乙锭(Ethidium monoazide bromide, EMA)与实时荧光定量PCR技术相结合, 建立一种能有效区分青枯菌死活细胞的检测方法。【方法】样品DNA制备前经EMA渗透预处理, 再进行实时荧光定量PCR特异扩增菌体DNA。【结果】终浓度为2.0 mg/L的EMA能有效排除1.0×107 CFU/mL灭活青枯菌细胞DNA的扩增, 对活细胞和不可培养状态(Viable but non-culturable, VBNC)活菌的DNA扩增均没有影响。当每个定量PCR反应体系中的活细胞在5.0×100?5.0×104 CFU范围内时, 扩增Ct值与定量PCR反应体系中活细胞CFU对数值呈良好的负相关性(R2=0.992 5)。比较EMA-qPCR法和平板计数法对经过不同温度短期保存的青枯菌检测结果发现, 待检样品可在24 °C与4 °C冷藏条件下短期保存。【结论】本研究建立的EMA-qPCR方法能有效检测青枯菌VBNC细胞和有效区分死活菌, 避免或减少青枯菌PCR检测的假阳性和假阴性。     

Elicitation of immunity in mice after immunization with the S2 subunit of the severe acute respiratory syndrome coronavirus

The S2 domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein is responsible for fusion between virus and target cell membranes, and is expected to be immungenic. In this study, we investigated the immune responses against the S2 subunit in BALB/c mice, which were vaccinated either with plasmid DNA encoding the S2 domain (residues 681-1120), the recombinant S2 fragment (residues 681-980) in incomplete Freund's adjuvant, or with inactivated SARS-CoV. The increased number of specific cytotoxic cells (CTLs) and the high titer of specific antibody showed stimulation of both arms of the immune system in these groups. The shift in cytokines suggested that Th1-polarized immune response was induced by plasmid pCoVS2, meanwhile the Th2-dominant response was induced by recombinant S2 fragment and inactivated vaccine. However, the titer of neutralizing antibodies was only detectable in mice immunized with inactivated virus, but not with pCoVS2 plasmid. Taken together, the S2 domain could induce specific cellular immune response and a high level of total IgG but little neutralizing antibodies against infection by SARSCoV.     

Ammonium accumulation is associated with senescence of rice leaves

The relationship between ammonium accumulation and senescence of detached rice leaves was investigated. Ammonium accumulation in detached rice leaves coincided closely with dark-induced senescence. Exogenous NH4Cl and methionine sulfoximine, which caused an accumulation of ammonium in detached rice leaves, promoted senescence. Treatments such as light and benzyladenine, which retarded senescence, decreased ammonium level in detached rice leaves. Abscisic acid, which promoted senescence, increased ammonium level in detached rice leaves. The current results suggest that ammonium accumulation may be involved in regulating senescence. Evidence was presented to show that ammonium accumulated in detached rice leaves increases tissue sensitivity to ethylene. The accumulation of ammonium in detached rice leaves during dark-induced senescence is attributed to a decrease in glutamine synthetase activity and an increase in reduction of nitrate.     

黄土丘陵区不同侵蚀环境下土壤有机碳对植被恢复的响应

植被恢复是提高土壤有机碳累积和储存的重要措施。以黄土丘陵区两个典型侵蚀环境下的小流域即黄土区坊塌流域和砒砂岩区满红沟流域退耕坡面为研究对象,分析了土壤有机碳含量(SOCC)、有机碳密度(SOCD)在同一侵蚀环境不同群落下的变化以及在同一群落不同侵蚀环境间的差异,旨在探明不同侵蚀环境下土壤有机碳对植被恢复的响应。结果表明:1)同一侵蚀环境下,与坡耕地相比,自然恢复方式下退耕地植被恢复初期SOCC、SOCD均显著降低,之后随植被恢复均显著升高(P0.05);人工恢复方式下退耕地20—25年柠条锦鸡儿群落和13—14年刺槐群落SOCC、SOCD均显著升高(P0.05),说明同一侵蚀环境内,退耕地在两种恢复方式下均能显著提高土壤有机碳累积和储存。2)同一侵蚀环境下,与相近恢复年限的自然恢复群落相比,刺槐群落SOCC、SOCD均显著高于长芒草+铁杆蒿群落(P0.05),砒砂岩区柠条锦鸡儿群落SOCC、SOCD均显著低于铁杆蒿群落(P0.05),黄土区柠条锦鸡儿群落SOCC显著低于而SOCD显著高于铁杆蒿群落(P0.05),说明相同恢复时间内,相对于自然恢复方式,人工刺槐造林在两种侵蚀环境下均能累积与储存较多的土壤有机碳,而柠条锦鸡儿造林在两种侵蚀环境下累积土壤有机碳的效果均不佳,在黄土区储存土壤有机碳效果好于砒砂岩区。3)同一群落下,黄土区人工和自然恢复群落SOCC均高于砒砂岩区;黄土区人工恢复群落SOCD均显著高于而自然恢复群落SOCD均低于砒砂岩区(P0.05),说明黄土区人工恢复累积和储存土壤有机碳及自然恢复累积土壤有机碳的效果较好,而砒砂岩区自然恢复储存土壤有机碳的效果较好。     

蚯蚓体内一种纤溶酶原激活剂(e-PA)的分离纯化

为获得一种高效,低廉的溶栓药物,从赤子爱胜蚓（Ｅｉｓｅｎｉａｆａｅｔｉｄａ）体内分离纯化出一种可体外激活纤溶酶原从而间接降解纤维蛋白的酶（ｅ－ＰＡ）．纯化过程包括：粗品的盐析,离子交换层析,凝胶过滤层析及疏水相互作用层析．该组份是由二个亚基通过疏水相互作用维系在一起的．通过凝胶过滤层析,可测得全酶的分子量为４５０００;ＳＤＳ电泳显示大、小亚基的分子量分别是２６０００与１８０００;而质谱法测得的大、小亚基的分子量分别为２４５５６．７与１５５４６．６．对大小亚基进行了氨基酸组成分析,结果显示大亚基不含Ｌｙｓ而小亚基不含Ｃｙｓ．测定了大亚基Ｎ端２５个氨基酸序列：ＶＩＧＧＴＮＡＳＰＧＥＩＰＷＱＬＳＱＱＲＱＳＧＳＷ．并与部分已知蛋白质序列进行了比较．ｅ－ＰＡ在纤维蛋白平板上表现有三种不同的纤溶活性     

乳杆菌DM9811挥发性脂肪酸组分对白色念珠菌、金黄色葡萄球菌的抑制

目的：探讨乳杆菌DM9811发酵液的挥发性脂肪酸对金黄色葡萄球菌、白色念珠菌抑制作用。方法：采用普通细菌计数法。结果：乳杆菌DM9811发酵液中挥发性脂肪酸对金黄色葡萄球菌、白色念珠菌具有抑制作用,以对白色念珠菌抑制作用最强,金黄色葡萄球菌抑制次之,两者都呈现剂量效应关系。结论：乳杆菌DM9811发酵液中挥发性脂肪酸对细菌、真菌都有明显的抑制作用。     

牛生长激素基因在马铃薯中的表达

将牛生长激素基因ｃＤＮＡ 与Ｐａｔａｔｉｎ ＣｌａｓｓＩ启动子及ＮＯＳ３终止子连接,构建了表达载体ｐＰＢＧＴ． 用直接法将表达载体转入农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍ ｅｆａｃｉｅｎｓ） ＬＢＡ４４０４（ｐＲＡＬ４４０４）菌株, 用此菌株转化马铃薯（Ｓｏｌａｎｕｍ ｔｕｂｅｒｏｓｕｍ ）得到再生植株． 经ＮＰＴⅡ活性检测,总ＤＮＡ ＰＣＲ和Ｓｏｕｔｈｅｒｎ ｂｌｏｔ证明目的基因已整合到马铃薯基因组中．ＲＮＡ 点杂交和Ｗｅｓｔｅｒｎ ｂｌｏｔ表明牛生长激素基因已在转基因马铃薯块茎中转录和表达     

理性设计盐桥构建伯克霍尔德菌脂肪酶热稳定突变体

【目的】借助蛋白质工程技术提高伯克霍尔德菌ZYB002脂肪酶LipA的热稳定性,以期更好地将其应用于工业生产中。【方法】利用YASARA、Fold X、Rosetta、Gromacs等生物信息学软件,构建1个脂肪酶Lip A的热稳定性提高的微型突变体电子文库;通过对突变体的结构信息和自由能变化进行评估,筛选出潜在的有价值的突变体。继而利用基因定点突变技术,构建上述候选突变体的突变基因文库,通过实验筛选出热稳定性提高的脂肪酶LipA突变体。【结果】利用上述方法,从构建的20个脂肪酶LipA突变体中,筛选到热稳定性有显著提高的3个突变体LipA-Asn~(125)Asp、LipA-Asn~(125)Glu和LipA-Gln~(262)Glu。经55°C处理12 min后,上述3个突变体的T1250值较野生型分别提高4.0°C、5.5°C和4.4°C;在55°C下的半衰期较野生型分别提高了2.2倍、3.8倍和2.6倍。【结论】利用生物信息学软件,构建脂肪酶LipA突变体电子文库,结合蛋白质的结构信息,可以快速筛选到热稳定性提高的脂肪酶LipA突变体。