Overexpression of malate dehydrogenase in transgenic tobacco leaves: enhanced malate synthesis and augmented Al-resistance

Numerous studies with transgenic plants have demonstrated that overexpression of enzymes related to organic acid metabolism under the control of CaMV 35S promoter increased organic acid exudation and Al-resistance. The synthesis of organic acids requires a large carbon skeleton supply from leaf photosynthesis. Thus, we produced transgenic tobacco overexpressing cytosolic malate dehydrogenase (MDH) cDNA from Arabidopsis thaliana (amdh) and the MDH gene from Escherichia coli (emdh), respectively, under the control of a leaf-specific light-inducible promoter (Rubisco small subunit promoter, PrbcS) in the present study. Our data indicated that an increase (120–130%) in MDH-specific activity in leaves led to an increase in malate content in the transgenic tobacco leaves and roots as well as a significant increase in root malate exudation compared with the WT plants under the acidic (pH 4.5) conditions irrespective of 300 μM Al³⁺ stress absence or presence. After being exposed to 25 μM Al³⁺ in a hydroponic solution, the transgenic plants exhibited stronger Al-tolerance than WT plants and the degree of A1 tolerance in the transgenic plants corresponded with the amount of malate secretion. When grown in an Al-stress perlite medium, the transgenic tobacco lines showed better growth than the WT plants. The results suggested that overexpression of MDH driven by the PrbcS promoter in transgenic plant leaves enhanced malate synthesis and improved Al-resistance.     

尼泊尔生态系统质量评估与时空格局变化——基于参照条件的评估方法

良好的生态系统质量是维持人类社会供给需求和可持续发展目标实现的重要保障。针对尼泊尔自然地理环境复杂多样，区域间气候差异明显的特点，结合基于参照条件的评估方法可以得到生态系统质量的相对水平值，其结果能够反映出不同的变化信息。植被是区域生态系统质量变化的重要指示器，利用尼泊尔五大地理区以及四种主要植被生态系统类型划分出20个生态评估区，从表征植被生态系统的水平结构、生产功能和垂直结构3个方面计算生态参数相对密度指标（RVI），结合主成分分析法构建植被生态系统质量指数（VEQI），并以其国家自然保护区为参照，构建基于参照条件的生态系统质量评估模型，计算了尼泊尔2016和2020年基于参照条件的植被生态系统质量指数（VEQI''）并分析其生态系统质量的时空格局变化。结果表明：（1）2016至2020年，尼泊尔生态系统质量现实值VEQI的平均值增加了3.49%，总体上，在参照生态系统质量（VEQI_ref）提高（约1.41%）的背景下，生态系统质量相对水平值VEQI''增加了1.42%；（2）对于尼泊尔地区，评估区89%分位数的VEQI与其对应的国家自然保护区的参照值具有很强的相关性，总体差异较小，可以代替作为参照值；（3）从空间格局变化趋势来看，尼泊尔生态系统质量变好、基本稳定和变差的面积分别占植被生态系统总面积的74.16%、14.25%和11.59%。与数量不足、较难收集利用的野外观测台站数据相比，国家自然保护区更接近理想参照生态系统的假设，通过有限的自然保护区确定生态评估区的参照值，实现生态系统质量的快速评估，其结果具有更好的时空可比性，可以为区域生态质量变化评估及量化分析等方面提供参考。     

Abundances and host relationships of chigger mites in Yunnan Province,China

This paper reports on ectoparasitic chigger mites found on small mammals in Yunnan Province, southwest China. Data were accumulated from 19 investigation sites (counties) between 2001 and 2009. A total of 10 222 small mammal hosts were captured and identified; these represented 62 species, 34 genera and 11 families in five orders. From the body surfaces of these 10 222 hosts, a total of 92 990 chigger mites were collected and identified microscopically. These represented 224 species, 22 genera and three subfamilies in the family Trombiculidae (Trombidiformes). Small mammals were commonly found to be infested by chigger mites and most host species harboured several species of mite. The species diversity of chigger mites in Yunnan was much higher than diversities reported previously in other provinces of China and in other countries. A single species of rodent, Eothenomys miletus (Rodentia: Cricetidae), carried 111 species of chigger mite, thus demonstrating the highest species diversity and heaviest mite infestation of all recorded hosts. This diversity is exceptional compared with that of other ectoparasites. Of the total 224 mite species, 21 species accounted for 82.2% of all mites counted. Two species acting as major vectors for scrub typhus (tsutsugamushi disease), Leptotrombidium scutellare and Leptotrombidium deliense, were identified as the dominant mite species in this sample. In addition to these two major vectors, 12 potential or suspected vector species were found. Most species of chigger mite had a wide range of hosts and low host specificity. For example, L. scutellare parasitized 30 species of host. The low host specificity of chigger mites may increase their probability of encountering humans, as well as their transmission of scrub typhus among different hosts. Hierarchical clustering analysis showed that similarities between different chigger mite communities on the 18 main species of small mammal host did not accord with the taxonomic affinity of the hosts. This suggests that the distribution of chigger mites may be strongly influenced by the environment in which hosts live.     

氮源NH4Cl浓度对粪产碱杆菌发酵生产热凝胶的影响

研究了利用粪产碱杆菌(Alcaligenes faecalis)发酵生产热凝胶的发酵条件,氮源是菌体生长的限制性底物,单纯地提高初始底物(氮源)浓度并不一定能促进细菌的生长和产物的合成.在分批发酵过程中,底物消耗导致培养环境pH的改变也是影响细菌进一步生长和产物合成的重要因素.通过增加培养基中初始氯化铵的浓度并同时控制发酵过程的pH条件,得到了较高的菌体浓度,热凝胶的合成水平也得到了显提高.当培养基中NH4Cl浓度提高到3.6g/L时,菌体浓度达到7.2g/L,热凝胶合成的产量可达30.5g/L,比原来NH4Cl浓度为1.1g/L时提高了51.7%.提高菌体浓度意味着需要提高溶氧水平来满足细菌的生长和代谢.初始氮源NH4Cl浓度的增加虽然能使菌体浓度得到提高,但发酵过程对溶氧的需求也相应增加,需要提高搅拌转速和通风以增加供氧水平.但高搅拌速率产生的高剪切力对热凝胶的凝胶性能将产生破坏作用,因此在发酵过程中需要综合考虑细菌培养密度对合成热凝胶产量和质量的影响.     

KcsA通道对Na+、K+及Rb+离子选择性的统计热力学研究

钾离了的通透率至少比钠离子的通透率大10000倍,这个问题至今没有很好地解决,为了存分子水平阐释钾离子通道的选择性机制,以KcsA钾通道X射线衍射结构为基础,采用密度泛函理论计算了不同离子在离子通道中的位能．计算结果表明,Rb^ 离子具有与K^ 离子相类似的位能曲线,但是其在通透过程遇到的位垒要比K^ 离子的位垒高,因而所对应的通透率也就小十钾离子的通透率,而钠离子的的通透率仅仅足钾离子通透率的0．0067％．文中所涉及的系统仪仅包含269个原子,而用分子动力学虽然也可以得到相近的结果,但是它的系统火小为41000个原子．     

抗人尿道高分化鳞癌单克隆抗体的研制

用本室建立的人尿道高分化鳞状上皮癌细胞株(Hus-98)免疫Balb/C小鼠,通过细胞杂交技术及ELISA方法进行筛选,SP免疫组织化学方法进行鉴定,获得10株能稳定分泌、选择性强、效价高的杂交瘤细胞株;同时从乳腺癌组织中提出肿瘤相关抗原行免疫印记实验(Western-blot)。结果6株经4次克隆能稳定分泌抗体Ig,免疫组化显示这些抗体是针对来源于上皮的鳞癌、腺癌细胞膜和细胞浆的,而癌旁正常组织及胎儿组织呈阴性反应;通过western-blot结果显示肿瘤相关抗原分子量为46.414kb。     

Ⅲ类内含子及其对基因表达的影响

内含子是指断裂基因中的非编码区序列。根据内含子的核苷酸序列和RNA潜在折叠方式不同,可分成四种类型：Ⅰ类内含子、Ⅱ类内含子、Ⅲ类内含子和Ⅳ类内含子。Ⅲ类内含子为真核细胞前mRNA中的内含子。它可以启动某些基因的表达,影响基因的表达量;增加mRNA分子的稳定性;并通过选择性剪接调控基因的表达。Ⅲ类内含子可以提高转基因动物基因的表达效率。因此,基因工程中,为了提高表达效率,是选用基因组基因还是cDNA基因,应视具体基因而定。     

应用水动力转染技术使人低密度脂蛋白受体、CD81和浸入诱导蛋白L同时在小鼠体内表达

为建立人低密度脂蛋白受体（LDLR）、CD81和浸入诱导蛋白L（Sip—L）等多个HCV感染相关分子共表达的转基因小鼠,首先分别构建了白蛋白启动子调控的人LDLR、CD81和Sip-L基因的小鼠肝脏组织特异性表达载体,将3种质粒同时采用水动力转染技术导入小鼠体内,FIT—PCR和免疫组化技术检测转入体内基因的表达及持续时间。结果表明,转染8h后即可检测到3种基因在体内转录,人LDLR和CD81在转染1～4d后可在50％-90％的肝细胞中高效表达,7d后检测不到目的基因表达。为了进一步延长转染基因在小鼠体内的表达时间,又分别构建了人LDLR、CD81和Sip—L的染色体整合型表达载体,将它们与噬菌体ФC31的整合酶表达载体同时水动力转染小鼠,3种基因在体内表达时间可持续到转染后25d。以上结果表明建立了多个HCV感染相关分子共表达的转基因小鼠,为确定这些分子能否使小鼠感染HCV奠定了基础。     

小鼠脂联素受体2基因编码区的克隆及序列分析

构建小鼠脂联素受体2(mAdipoR2)基因cDNA克隆,并进行序列及基因结构分析,为进一步研究小鼠AdipoR2的表达和生物学活性奠定基础。用RT—PCR方法扩增mAdipoR2基因cDNA,获得的片段连接至pGEM-T载体,转化JM109大肠杆菌,经酶切鉴定后,进行序列测定。利用因特网生物信息学资源分析mAdipoR2基因序列。结果成功地构建了mAdipoR2基因cDNA克隆,其序列与GenBank登录序列一致。通过与小鼠基因组序列比对,分析了mAdipoR2基因结构,其编码序列由7个外显子组成,编码1个含7个跨膜区的膜蛋白,但该受体不属于G蛋白偶联受体家族(GPCRs)。mAdipoR2与人及大鼠蛋白的同源性分别为91％和95％。     

小鼠脂联素与绿色荧光蛋白融合基因表达载体的构建及在COS-7细胞中的表达

以绿色荧光蛋白(GFP)基因(gfp)为报告基因,构建小鼠脂联素(mADPN)基因(mAd)与gfp的融合基因mAd/gfp表达载体pCI-neo-apoEHCR-hAATp-mAd-gfp,脂质体法转染体外培养的COS-7细胞,荧光显微镜观察GFP在细胞中的表达可间接反映mADPN的表达,并通过RT-PCR在核酸水平进一步确证mAd的表达.荧光显微镜观察及RT-PCR结果均证明mADPN在COS-7细胞中获得了高效表达,表明mADPN重组表达载体pCI-neo-apoEHCR-hAATp-mAd可以在真核细胞COS-7中高效表达mADPN,为进一步探讨mAd在小鼠体内的表达提供了可行性依据.