一株重组大肠杆菌/pET-28a-lpgad的构建及其高效生产γ-氨基丁酸转化条件的优化

为实现微生物法高效率生产γ-氨基丁酸(GABA),从一株经多次诱变筛选的具有较高谷氨酸脱羧酶(GAD)活力植物乳杆菌GB 01-21全基因组DNA中PCR扩增获得GAD酶基因lpgad,构建重组质粒pET-28a-lpgad,在大肠杆菌E.coli BL21(DE3)中高效诱导表达。并采用Ni柱亲和层析纯化获得重组GAD,并对其酶学性质进行初步研究,为改良转化工艺提高GABA产量提供可靠理论依据。结果显示,重组大肠杆菌中GAD酶活显著提高,可达8.53 U/mg,是植物乳杆菌GB 01-21中GAD酶活的4.24倍。将该重组菌应用于转化L-谷氨酸生产GABA,5 L发酵罐水平转化24 h产量可达143.5 g/L,摩尔转化率为97.32%,是植物乳杆菌GB 01-21的2.19倍。纯化后酶学性质进行初步研究表明:其最适pH为4.8;最适温度为37℃;Ca2+、Mg2+对其有较强的激活作用,将上述实验结果用于转化条件的优化,最终5 L发酵罐上进行转化实验,批次添加底物L-谷氨酸共600 g,转化24 h,GABA累计浓度可达204.5 g/L,摩尔转化率为97.92%,与最初转化条件相比,GABA浓度提高了42.5%,为其工业化应用打下了良好的基础。     

地带性森林群落物种多样性的比较研究

在群落调查以及文献收集的基础上,对暖温带地区、暖温带和亚热带过渡地区、亚热带地区以及热带地区的典型地带笥植被不同群落类型群落多样性进行了分析和研究。结果发现这４类地区物种多样性的变化具有一定的规律性,具体表现在热带地区群落乔木层的物种丰富度和多样性为最高,然后依次是亚热带地区、秦蛉地区和东灵山地区;灌木层的物种丰富度和多样性则以亚热带地区为最高,其次是秦岭地区,热带地区的则仍高于东灵山地区;灌木层     

微小亚历山大藻麻痹性贝类毒素小鼠生物检测

应用小鼠生物检测法对培养的微小亚历山大藻（LJX02株系）麻痹性贝类毒素进行了检测。首先建立微小．[X02亚历山大藻培养技术,利用对数期藻细胞接种,可使藻细胞处于快速生长,并在7d内藻细胞密度最高达到30000ml^-1。从培养株系中提取的藻毒素,经液相质谱分析主要为GTX1／4和GTX2／3,其含量分别为12．23μg／ml和9．742μg／ml,12L藻体培养液中可获得18．35μgGTX1／4和14．61μgGTX2／3毒素量。采用小鼠生物检测法建立了藻毒素剂量-小鼠死亡时间曲线,其曲线方程为Y=74．017X^-1.5896,将死亡时间t变为时间倒数1／t,GTX毒性MU取对数为logMU,得直线回归方程为Y=3．7009X-0．0858（R^2=0．9824）,求得了昆明小鼠一个鼠单位所代表的GTX1／4和GTX2／3毒素剂量分别为0．026μg和0．021μg。     

灵芝栽培培养基筛选试验

以灵芝(Ganoderma lucidum)之赤芝(Ganoderma lucidum Karst)、韩芝(Ganoderma luci- dum H.G)为试验品种,培养基以棉壳98%、石膏1%、蔗糖1%为对照,另外以棉壳、麸皮、木屑、氨基酸不同含量配制4种培养基作比较栽培试验。结果表明:2号培养基出菇产量高、菇形好,对赤芝、韩芝都适宜;赤芝、韩芝对培养基的要求略有差异,韩芝在含麸皮量高的基质上栽培更为适合。     

Disruption of gene SPL35, encoding a novel CUE domain‐containing protein,leads to cell death and enhanced disease response in rice

Lesion mimic mutants that exhibit spontaneous hypersensitive response (HR)‐like necrotic lesions are ideal experimental systems for elucidating molecular mechanisms involved in plant cell death and defence responses. Here we report identification of a rice lesion mimic mutant, spotted leaf 35 (spl35), and cloning of the causal gene by TAIL‐PCR strategy. spl35 exhibited decreased chlorophyll content, higher accumulation of H₂O₂, up‐regulated expression of defence‐related marker genes, and enhanced resistance to both fungal and bacterial pathogens of rice. The SPL35 gene encodes a novel CUE (coupling of ubiquitin conjugation to ER degradation) domain‐containing protein that is predominantly localized in cytosol, ER and unknown punctate compartment(s). SPL35 is constitutively expressed in all organs, and both overexpression and knockdown of SPL35 cause the lesion mimic phenotype. SPL35 directly interacts with the E2 protein OsUBC5a and the coatomer subunit delta proteins Delta‐COP1 and Delta‐COP2 through the CUE domain, and down‐regulation of these interacting proteins also cause development of HR‐like lesions resembling those in spl35 and activation of defence responses, indicating that SPL35 may be involved in the ubiquitination and vesicular trafficking pathways. Our findings provide insight into a role of SPL35 in regulating cell death and defence response in plants.     

Glutamate receptor,ionotropic, N-methyl D-aspartate-associated protein 1, a potential target of miR-296, facilitates proliferation and migration of rectal cancer cells

ABSTRACT

The purpose of our article was to probe the influence of GRINA on rectal cancer and how GRINA is regulated in rectal cancer. Based on the public data, we found that GRINA was highly expressed in rectal cancer tissues and related to worse prognosis in rectal cancer patients. MiR-296 was predicted as an upstream regulatory miRNA of GRINA, which was further verified by dual-luciferase reporter assay. Moreover, we revealed that up-regulation/down-regulation of GRINA facilitated/suppressed SW1463/SW837 cell proliferation, migration, and invasion. Rescue assays indicated that the facilitating impact of GRINA on SW1463 cell proliferation and motility was abolished by miR-296 over-expression whilst the suppressing influence of GRINA on SW837 cell proliferation, migration, and invasion was reversed by miR-296 depletion. These consequences indicated that GRINA, which might be regulated by miR-296, acted stimulative important impact on rectal cancer cells, insinuating that GRINA might be a novel potential target for rectal cancer therapy.