人新基因hbrp的染色体定位及其对TPK活性的抑制作用（简报）

hbrp(Human BSP-Related Protein)是我们实验室最近在睾丸组织中克隆的一个人与BSP（bovine seminal plasma)蛋白相关的新基因。为了将有关该新基因信息与现有人类基因组转录图相整合,我们应用荧光原位杂交(fluorescent in situ hybridizationFISH)法进行了该基因的人染色体基因定位,结果成功地将hbrp基因定位在人19号染色体长臂1区3带上。hbrp基因是在对BSP蛋白功能的研究过程中发现并克隆的,其同源性分析发现,与其序列最相近     

GFP-mTn融合蛋白对蓝猪耳生活细胞微丝骨架的标记

用农杆菌介导法将嵌合基因GFP-mTn(mTn是微丝结合蛋白Talin的微丝结合域,可以显示活体细胞中微丝的结构)导入蓝猪耳。经激光共聚焦显微镜观察了转基因植株的各种不同组织中融合蛋白的表达和分布情况。在叶片的表皮细胞、保卫细胞、根部的皮层细胞中有融合蛋白的不同程度表达。但仅在保卫细胞中微丝标记状况良好,显示基因表达的组织特异性。经光诱导处于开放态的气孔的保卫细胞微丝呈网状结构,在细胞内无规则分布;经黑暗诱导处于关闭态的气孔保卫细胞中微丝束沿保卫细胞纵轴排列,呈卷曲状分布,并观察到螺旋和环状的微丝结构。在转基因植株的其他部位,例如茎表皮细胞、根毛细胞和花粉粒中,未检测到目的基因的表达。本研究获得的转基因植株为研究气孔运动过程中微丝动态变化提供了有用的材料。     

中国人胃癌染色体17q21区域基因遗传不稳定性研究

To investigate microsatellite instability (MSI) and loss of heterozygosity (LOH) of locus D17S396, D17S579 and D17S855, and their effect on the expression of nm23H1 and BRCA1 of gastric cancer, which would provide experimental basis for clinical treatment and prognosis analysis of gastric cancer. DNA was extracted from paraffin-embedded materials. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used to analyze MSI and LOH. Expression of nm23H, and BRCA1 was detected by Envision immuno-histochemistry and Leica-Qwin computer imaging techniques. In the forty cases of gastric cancer, the frequency of MSI, LOH and nm23H1 protein were 20.00%, 17.50% and 55.00% respectively at locus D17S396, while at locus D17S579, the frequency of MSI, LOH and BRCA1 protein were 22.50%, 15.00% and 37.50% respectively; at locus D17S855, the frequency of MSI, LOH and BRCA1 of thirty-seven cases were 18.92%, 18.92%, 37.84% respectively. In tumor node metastasis (TNM) staging, at locus D17S396, D17S579 and D17S855, MSI in stages I + II appeared more frequently than that in stages III + IV, while LOH appeared the contrary tendency. In the group of metastasis of gastric cancer, MSI had a less frequency (5.00%) than that with no metastasis (35.00%, P < 0.05) at locus D17S396, but LOH appeared more frequently (30.00%) than that with no metastasis (5.00%, P < 0.05). At locus D17S579, MSI had an increasing tendency with the degree of tumor differentiation (50.00% in high differentiation cases, 20.00% in middle differentiation cases, and 0% in low differentiation cases, P < 0.05). The frequency of nm23H1 and BRCA1 protein in stages TNM I + II was higher than that in stages TNM III + IV; and that in higher differentiation cases was higher than in poor differentiation cases. The frequency of nm23H1 protein in the group of metastasis (30.00%) was less than that with no metastasis significantly (80.00%, P<0.01). The frequency of nm23H1 protein in the group positive to MSI (87.50%) was higher than that in the group negative to MSI (46.88%, P < 0.05). However, nm23H1 protein in group positive to LOH (14.29%) was lower than that in the group negative to LOH (63.64%, P < 0.05). The frequency of BRCA1 protein in the group positive to MSI (66.67%) was more than that in the group negative to MSI (29.03%, P < 0.05). The results of experiments indicate that MSI and LOH may separately control the development of sporadic colon cancer with different pathways. MSI may be an early period molecule marker for sporadic colon cancer, enhanced expression of nm23H1 protein can effectively inhibit colon cancer metastasis and improve prognosis of sporadic colon cancer patients. By comparison, LOH mostly arises in the late period of sporadic colon cancer and endows a high aggressive and poor prognostic phenotype. nm23H1 protein could effectively restrain gastric cancer metastasis and development; and BRCA1 protein could restain tumor from becoming lower differentiation.     

奶山羊转基因供核细胞的再饥饿对核移植胚胎发育的影响

为提高转基因奶山羊体细胞核移植胚胎早期发育率,将经转染外源基因的山羊胎儿成纤维细胞经饥饿培养(含0．5％FCS的DMEM)5天后分成两部分：第一部分细胞-80℃或液氮冻存,试验前复苏后直接用作供核细胞(试验组Ⅰ),或复苏后恢复培养(含10％FCS的DMEM)2-5天后再饥饿5天用作供核细胞(试验组Ⅱ);第二部分细胞作传代培养(含10％FCS的DMEM)2天后再饥饿5天用作供核细胞(试验组Ⅲ)。将上述不同处理的供核细胞进行细胞周期与存活率的检测,并将该供核细胞移入去除遗传物质的山羊MⅡ期卵母细胞的卵周隙内,经电融合、化学激活后,将核移植(NT)胚胎经0．8％琼脂糖包理后移入临时寄母输卵管内,培养6天后回收并观察NT胚胎的早期发育。结果,试验组Ⅱ所用供核细胞中G0／G1期细胞所占比例及其存活率分别为95．68％、99．9％,均显著地高于试验组Ⅰ(88、66％、80％);试验组Ⅱ的桑椹及囊胚期NT胚胎的发育率(66．09％)显著地高于试验组Ⅰ(22．00％)与试验组Ⅲ(50．5l％)。将以上发育的NT胚胎分别移入同步发情的受体后,35天作B超妊娠诊断,试验组Ⅱ的受体妊娠率为45．83％,显著地高于试验组Ⅰ(20．00％)与试验组Ⅲ(29．58％)。流式细胞仪分析结果表明,饥饿后的供核细胞经冷冻,复苏后恢复培养2-5天,再经饥饿处理,能显著地提高G0／G1期细胞的比例及细胞存活率;应用该细胞所组建的NT胚不仅具有较高的桑椹与囊胚期发育率,而且具有较高的受体妊娠率。     

小鼠α1，2岩藻糖基转移酶基因的克隆及表达

本文报告小鼠GDP岩藻糖：β半乳糖苷α1,2-岩藻糖基转移酶（α1,2-fucosyltansferase,α1,2-FT）基因的克隆,并进行功能鉴定。利用RT—PCR方法克隆小鼠α1,2-岩藻糖基转移酶基因编码区MFUT-II,测序后将其插入表达载体pcDNA3．1的多克隆位点,构建表达载体pcDNA3．1-MFUT-II;采用磷酸钙法将其转染于COS-7细胞进行表达,通过对底物特异性比较研究酶的性质;应用Northern印迹杂交法研究基因在小鼠组织中的表达情况;应用Southern印迹杂交法分析基因存在状态。结果证实MFUT-II为小鼠α1,2-岩藻糖基转移酶基因家族的新成员。含有一个完整的开放读码框。可编码347个氨基酸。其估计分子质量为39kDa,和小鼠日及Sec1基因具有序列同源性。分别与人类Se基因（79．O％）、大鼠Ratrrs（89％）基因、兔Rabbit FT-III基因（77％）具有较高的序列同源性。用MFUT-II基因转染的COS-7细胞具有α1,2-FT活性。MFUT-II可在多种组织中产生3．5kb大小的mRNA转录产物。基因Southern印迹杂交分析结果显示：基因MFUT-II仅为一个拷贝。这些结果证明MFUT-II为小鼠的Se基因。     

苹果Co基因的SSR标记定位（简报）

Columnar apple is an important genetic resource for tree form breeding of apple. In this study, 106 individuals of the F1 population derived from 'Spur Fuji' (coco)x 'Telamon'(Coco) were used as plant materials for screening SSR markers linked to gene. By bulked segregating analysis (BSA), eight SSR markers from the tenth linkage group of apple genome were tested. Finally, three of them, COL, CH02a10 and CH03d11, were identified to be SSR markers of Co gene. Linkage analysis showed that the genetic distance of COL, CH02a10 and CH03d11 to Co locus was 15.3cM, 22.2cM and 3.9 cM, respectively. On the linkage map of these markers, Co gene was located between COL and CH03d11.     

影响小鼠体细胞脂质体法转染效率的因素

We studied the effects of the amount of liposome and plasmid, exposure time of cells to the liposome-plasmid complexes, number of cell passages and cell types on GFP gene transfection of mouse somatic cells. The maximal GFP transgene expression (30.7%) was achieved when mouse fetal fibroblast cells (MFFC) at 70%-90% confluence of passage 3 were exposed for 6 h to the complexes of 4 microg liposome (LipofectAMINE) and 0.3 microg plasmid (pEGFP-N1). Under these conditions, we compared the effect of the number (from primary to 15) of passages on the transfection efficiency of MFFC. The transfection efficiency of MFFC was 10.0%, 28.9% and 7.2% at the primary, 3rd and 15th passage, respectively, which indicated that the transfection efficiency decreased with passaging. When MFFC, mouse oviductal epithelial cells (MOEC) and mouse granulosa cells (MGC) were transfected at passage 3, the transfection efficiency was 27.8%, 13.7% and 14.2%, respectively, under the described transfection conditions. When the cell cycle stages of different cell types at transfection were examined, it was found that 17.2% of MFFC, 8.7% of MOEC and 9.9% of MGC were at M phases of the cell cycle. Examination of the cell cycle stages of MFFC at different passages showed that MFFC at the third passage had the highest percentage of M cells and the percentage decreased afterwards. This suggested that the transfection efficiency was correlated with the percentages of cells at M phase, and provided essential data for improvement of the transfection efficiency.     

三江源地区不同植被土壤固氮微生物的群落结构研究

利用PCR_RFLP和测序分析法对位于青藏高原腹地三江源自然保护区的高寒草甸、高寒草原和高山森林等不同植被类型的土壤固氮微生物的群落组成进行了探讨。经过PCR_RFLP分析固氮基因nifH ,在3个样品中共得到2 33个克隆和99个可操作分类单元(OTUs) ,NQ_1样地具有最多的克隆数和OTUs,多样性为4 9 74 % ,在所有样品中分别具有1～2个明显的优势种群(占总克隆数>15 % ) ,并且具有4个共同的OTUs。选取了2 6个克隆进行基因测序分析,通过DNAMAN比较表明,这些序列间具有6 6 %～98%的相似性,并且在GenBank数据库中没有发现完全匹配的序列,因此这些序列可能代表着新的固氮生物株系。最后利用ClustalW与Mega软件构建了系统发育树,结果发现,这些序列被分为4个不同的簇,部分序列与属于蛋白细菌(Proteobacteria)的已知细菌具有近的亲缘关系,但是更多的序列与已知细菌具有较远的亲缘关系,而且nifH基因序列的分布在样地间没有明显的聚类     

鼠伤寒沙门氏菌SL7207SifA-突变株的构建和鉴定

鼠伤寒沙门氏菌SifA-基因突变株的特点是能进入真核细胞的胞液。利用P22噬菌体转导技术构建了鼠伤寒沙门氏菌疫苗株SL7207的SifA-突变株SL7207*,该突变株与SL7207有着相似的体外生长曲线和细胞侵袭力,SL7207*在MDCK上皮细胞中的增殖能力增强,但在RAW2647巨噬细胞中的生存能力减弱。小鼠毒力试验显示SL7207*在BALB/c小鼠体内毒力下降。仅SL7207*在体外可向RAW2647巨噬细胞递送真核表达质粒。SL7207*的构建为重组沙门氏菌疫苗载体的研制提供了一个新的选择。     

养殖牡蛎体内检出坎氏弧菌的鉴定

2 0 0 3年 9月从福建近海养殖太平洋牡蛎 (Crassostreagigas)体内分离出 4株细菌 ,对它们形态、生理生化特征、盐度、温度和pH生长条件以及 16SrRNA基因序列进行了研究。结果表明 ,4株细菌均为革兰氏阴性杆菌 ,具极生单鞭毛 ,发酵葡萄糖产酸不产气 ,氧化酶阳性 ,生长需NaCl,无色素 ,不发光 ,在TCBS平板上形成中等大小圆形绿色菌落 ,对弧菌抑制剂O 12 9敏感 ,具有弧菌属的典型特征。结合 16SrRNA基因序列分析结果 ,该菌 (编号SXS1)与坎氏弧菌的亲缘关系最为接近 ,其同源性达 99 .0 % ,因此将该菌鉴定为坎氏弧菌。对该菌在环境中的分布与水产养殖动物疾病的关系进行了讨论。