1株聚乳酸降解细菌的筛选、鉴定及产酶研究

从污泥中筛选出1株对聚乳酸（poly—L—lacticacid,PLA）具有降解活力的细菌DSL09,该菌株对PLA的乳化液、粉末及薄膜都具有降解作用。通过形态学、16SrDNA比对及生理生化特性的分析,鉴定该菌株属于芽胞杆菌属（Bacillus sp．）。为提高该菌株对PLA的降解活力,对其进行了紫外诱变,获得了稳定遗传的突变株DSL09-60b,该突变株的PLA降解活性提高至原始菌株的1．5倍。对该突变株产PLA降解酶的发酵条件进行了优化,经测定DSL09-60b在初始培养基pH为8．0、0．5％酪蛋白为诱导物、接种量6％（体积比）的条件下37℃培养54h时发酵液酶活性最高。     

Potential role of ABCA7 in cellular lipid efflux to apoA-I

ABCA7 is homologous to ABCA1 and has recently been shown in cell culture to bind apolipoprotein A-I (apoA-I) and to promote the efflux of phospholipids. However, it is not known if ABCA7 promotes lipid efflux in vivo. When expressed in HEK293 cells, both human and mouse ABCA7 promoted phospholipid efflux to apoA-I but no detectable cholesterol efflux. However, genetic knockdown of ABCA7 in mouse peritoneal macrophages did not affect phospholipid or cholesterol efflux to apoA-I. Moreover, in ABCA1-knockout macrophages, there was no detectable apoA-I-stimulated phospholipid efflux, inconsistent with a residual role of ABCA7. In contrast to plasma membrane localization of ABCA7 in transfected embryonic kidney cells, immunofluorescence microscopy of endogenous ABCA7 in macrophages showed a predominantly intracellular localization of the protein. Strikingly, immunofluorescence studies of adult mouse kidney revealed an apical brush border membrane localization of ABCA7 in the proximal tubule, suggesting that ABCA7 may come in contact with apoA-I in the glomerular filtrate. Although ABCA7 does not contribute to apolipoprotein-mediated lipid efflux in resting macrophages, its cell surface location in the kidney suggests that it could serve such a role in tissue microenvironments.     

用彗星实验技术分析MTX对小鼠细胞DNA的损伤作用

MTX是一种抗叶酸药物 ,作用于增殖细胞 ,为了解其作用机制和探测其遗传毒性靶器官 ,以小鼠为研究对象 ,用彗星实验技术检测了MTX腹腔注射染毒后对脾、骨髓、胸腺、和外周血淋巴细胞的DNA损伤作用及其与MTX剂量间的相关。 1.2 5～ 5mg/kgMTX可诱发小鼠体内 4种细胞的DNA单链断裂 ,核DNA损伤程度与用药剂量呈正相关。不同种类细胞对MTX的易感性不同 ,脾、骨髓、胸腺、外周血淋巴细胞可能是MTX的遗传毒性靶细胞。外周血淋巴细胞在SCGE分析中的拖尾现象可作为用药后组织器官对药物敏感性反映的生物标志     

铜绿假单胞菌体外对肠上皮细胞骨架的影响

为探索铜绿假单胞菌粘附肠上皮细胞后,细胞骨架特性的变化规律及可能的机制。采用微管吸吮实验技术并结合细胞ELISA、图像分析等方法研究体外绿脓杆菌粘附肠上皮细胞后细胞骨架的变化。结果显示细菌粘附后1h肠上皮细胞活力即开始下降,3h后IEC面积明显减小,而细胞周长无明显变化;胞内骨架蛋白减少,且随孵育时间的延长愈趋明显;细胞弹性系数K1、K2在粘附后3h明显降低,同时伴有粘性系数μ也明显下降。以上结果表明绿脓杆菌粘附肠上皮细胞后,细胞骨架成分改变,细胞骨架功能受损害,最终导致细胞损伤。     

钙调素对细胞周期的调节

RC3细胞是一种用真核表达载体1~(CaM)转染NIH 3T3细胞建成的可调钙凋素(Calmodulin,CaM)高表达细胞模型。通过分子杂交及蛋白免疫印迹方法证实在地塞米松(Dexamethasome,DXM)作用下,RC3细胞可高表达CaM。CaM的过表达使G_1期细胞减少,S期细胞增加;CaM拮抗剂三氟拉嗪(trifluoperazine,TFP)则使G_1期细胞增加,S期细胞减少。高表达CaM使细胞分裂指数提高,G_2期细胞减少,有丝分裂前期细胞增加,M中期细胞比例下降。而TFP处理则使分裂指数下降,G_2期细胞增加,M前期细胞减少,M中期细胞增加。实验结果表明CaM在G_1/S、G_2/M和M中期/M后期3个位点上对细胞周期进行调控;通过加速G_1至S期,G_2至M期和M中期至M后期的进程,使细胞倍增时间缩短,促进细胞增殖。本工作表明,RC3细胞作为CaM表达可调细胞模型,是研究细胞周期调控的有力工具。     

紫菜外生细菌抑菌活性及其多聚酮合酶(PKS Ⅰ)基因筛选

[目的]基于紫菜外生细菌抑菌活性的研究,本文对具有广谱抑菌活性的菌株进行了多聚酮合酶(Polyketide synthase Ⅰ,PKS Ⅰ)基因的筛选,以期获得PKS Ⅰ阳性菌株及探讨紫菜藻际微生物区系细菌的拮抗机制与PKS Ⅰ途径的关系.[方法]利用琼脂柱法筛选出具广谱抑菌活性的菌株31株,以其基因组DNA为模板,设计引物扩增酮基合成酶(Ketosynthase, KS)片段基因并将其克隆到pMD19-T Vector,筛选出PKS Ⅰ阳性菌,进行16S rDNA测序分析.[结果]紫菜外生细菌表现出广谱抑菌性.从温州病烂紫菜外生菌中筛选出3株具强抑菌活性的PKS Ⅰ阳性菌,BLAST比对结果显示:菌株WPhG3、WPySw1和WPySw2扩增得到PKS Ⅰ的KS结构域核苷酸序列所对应的氨基酸序列与Bacillus subtilis subsp. subtilis str. 168(NP. 389602)、Bacillussubtilis (ABR19776)和Aspergillus carbonarius(AAZ99721)的PKS Ⅰ的KS结构域的同源性分别达到98%、99%和98%;16S rDNA系统发生学分析显示它们均与Bacillus的同源性最高.[结论]紫菜藻际微生物群落组成复杂,通过多条途径调节藻际微生物区系的平衡.PKS Ⅰ途径可能是温州病烂紫菜外生菌Bacillus表现抑菌活性的一种方式.     

溶液流动对等电溶菌酶晶体生长的影响

本文对等温自由生长和强制性溶液生长的等电溶菌酶的晶体形态进行了研究,发现这些形态变化与溶液相的流动密切相关,指出生物晶体生长停止是由于生长晶体周围的溶质贫乏造成的;通过某些手段减薄或消除这一溶质贫乏区,就可以保证晶体的持续生长。本文的研究对改善大尺寸晶体的生长提供了一条途径。     

天麻栽培用蜜环菌的遗传多样性、生长和水解酶

蜜环菌Armillaria是一种广泛分布的担子菌,是栽培中药天麻所必须的共生菌。为揭示我国天麻栽培用和鄂西地区部分野生蜜环菌资源的遗传多样性和生物学特性,本文收集了9个省市EF-1-α的19个蜜环菌商品菌株和鄂西地区20株野生蜜环菌菌株,以IGS、ITS和elongation factor-1-alpha（EF-1-α）为分子标记对这些菌株进行系统发育与遗传多样性分析,并对影响与天麻共生的生物学特性（菌索发育和胞外水解酶活性）进行对比研究。结果表明,这19株蜜环菌商品菌株分别归属于2个clade。其中,有18个菌株聚在cladeⅢ,说明不同来源的蜜环菌商品菌株之间系统发育关系很近;通过对蜜环菌的系统发育、菌索生长以及胞外水解酶活性的研究,总体上商品种优于鄂西的野生蜜环菌生物种。从鄂西野生蜜环菌中筛选出4个具有潜在栽培价值的菌株。本研究为天麻栽培生产中蜜环菌菌种的选择提供参考。     

Dissecting the Genetic Basis of Extremely Large Grain Shape in Rice Cultivar ＇JZ1560＇

Rice grain shape,grain length(GL),width(GW),thickness(GT)and length-to-width ratio(LWR),are usually controlled by multiple quantitative trait locus(QTL).To elucidate the genetic basis of extremely large grain shape,QTL analysis was performed using an F2 population derived from a cross between a japonica cultivar ’JZ1560’(extremely large grain)and a contrasting indica cultivar ’FAZ1’(small grain).A total number of 24 QTLs were detected on seven different chromosomes.QTLs for GL,GW,GT and LWR explained 11.6%,95.62%,91.5%and 89.9%of total phenotypic variation,respectively.Many QTLs pleiotropically controlled different grain traits,contributing complex traits correlation.GW2 and qSW5/GW5,which have been cloned previously to control GW,showed similar chromosomal locations with qGW2-I/qGT2-I/qLWR2-2 and qGW5-2/qLWR5-l and should be the right candidate genes.Plants pyramiding GW2 and qSW5/GW5 showed a significant increase in GW compared with those carrying one of the two major QTLs.Furthermore,no significant QTL interaction was observed between GW2 and qSW5/GW5.These results suggested that GW2 and qSW5/GW5 might work in independent pathways to regulate grain traits.’JZ1560’ alleles underlying all QTLs contributed an increase in GW and GT and the accumulation of additive effects generates the extremely large grain shape in ’JZ1560’.