大头蛙Sox基因的克隆与序列分析

参考人SRY基因HMC—box的保守区序列,设计一对特异引物,采用PCR技术扩增了大头蛙的Sox基因,并对扩增产物进行了克隆与测序。结果在雌雄个体中均扩增出217bp的基因片段,与人对照相同。序列分析表明,大头蛙雌雄个体之间Sox序列没有差异,与人SOX基因的同源性达到88％,与其它各类动物的Sox基因也都有非常高的相似性。结果支持Sox基因在进化上十分保守的结论。     

九香虫(椿象)体内可培养细菌的多样性分析

【目的】认识药用昆虫九香虫(Aspongopus chinesis Dallas)成虫体内可培养细菌资源多样性。【方法】运用纯培养法、反转录重复因子扩增(BOXA1R-PCR)分析技术、16S r RNA基因测序和系统发育分析对样品中可培养细菌进行多样性研究,测定了分离菌株的抗菌特性、吲哚乙酸(IAA)含量和产淀粉酶活性等指标。【结果】通过6种不同培养基共分离得到52株菌落特征不同的细菌菌株。基于菌落特征和BOXA1R-PCR图谱选取12株代表菌株用于16S r RNA基因序列测定。16S r RNA基因序列系统发育分析显示,52株菌株分属于芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)、寡养单胞菌属(Stenotrophomonas)和伯克霍尔德氏菌属(Burkholderia)4个属,其中芽孢杆菌属(Bacillus)为优势菌属。分离到的52株细菌有44株(占总分离菌株的84.6%)表现出对供试病原菌具有较好的抑制作用,高达94.2%的分离菌株能产IAA,有43株(占总分离菌株的82.7%)表现出淀粉酶活性。【结论】九香虫内细菌种群较为多样,具有潜在应用价值。     

Ecdysteroid as a mediator in the regulation of silk protein synthesis and its influence on silkworm (Bom., Lepidoptera) genome

Abstract:  Ecdysteroid is one of the two most important insect hormones, which controls growth and developmental activities of insect. In silkworm, Bombyx mori L., basically ecdysteroid induces molting and metamorphosis in larvae and exogenous administration hastens the silk synthetic activity and cocoon spinning process but it strictly depends on the time of administration. In the present study, we administered phytogenous ecdysteroid to the 48-h-old 5th instar silkworm, B. mori at a single dose of 2  μ g per larva to study its effects on the larval growth, cocoon and silk variables. The possible role of ecdysteroid in altering the developmental sequence of silkworm and thereby its effect on cocoon and silk production at molecular level have been tried to get elucidated. The genomic DNA was isolated from the posterior silk gland on day 5 and 7 of the 5th instar from the ecdysteroid treated and the control larvae and was randomly amplified with arbitrary primers. The result presented notable variation in the amplified product suggesting the participation of ecdysteroid in regulating the silk gene. The feeding period of treated larvae was unaffected while the cocoon characters exhibited considerable improvement. The filament traits also were improved notably in the treated larvae. The participation of ecdysteroid in the silk biosynthetic process with its physiological and molecular implications was discussed.     

利用定点突变分析白念珠菌依赖铁基因FRP1启动子元件

通过对白念珠菌高铁还原酶基因FRP1启动子进行突变分析, 确认启动子中特殊调控元件。我们通过分析FRP1起始密码子上游1000 bp序列发现在-160 和-650处有2个推测的Rim101p结合位点, 对其分别进行定点突变, 然后构建启动子与报告基因LacZ融合质粒, 转化整合到白念珠菌rim101-/-株和野生株中, 检测不同缺铁条件下b-半乳糖苷酶活性。结果发现碱性条件, Rim101p能够正向调控FRP1的表达; 启动子-160处突变对启动子功能影响较弱, 而-650突变使启动子活性大大降低, 此结果和双突变的结果相同, 表明Rim101p主要通过与启动子-650处结合位点相互作用来调控FRP1的表达。     

表皮生长因子和降钙素基因相关肽在高频电磁场 治疗急性胃损伤中作用研究

目的：探讨高频电磁场（high-frequency electromagnetic fields,HEMFs）曝露治疗急性胃损伤的作用机制。方法：选用健康SD大鼠,以Indomethacin灌胃法复制胃粘膜急性损伤模型,观察40．68MHz HEMFs（波长为7．3m）曝露（微热量,30—50mA,15min,1／d）治疗1或6次后,大鼠胃粘膜损伤程度（胃损伤指数和病理损伤积分）、胃粘膜血流量、血浆表皮生长因子（epidermal growth factor,EGF）和降钙素基因相关肽（calcitonin gene—related peptide,CGRP）水平。结果：HEMFs曝露1次后,胃粘膜血流量较对照组明显升高（P〈0．05）,大鼠胃粘膜损伤程度、血浆EGF和CGRP水平较对照组均无显著性差异（P均〉0．05）;HEMFs曝露6次后,胃粘膜损伤程度较对照组明显改善（P均〈0．05）,胃粘膜血流量显著升高（P〈0．05）,血浆EGF和CGRP水平较对照组无显著性差异（P均〉0.05）。结论：HEMFs曝露治疗大鼠胃粘膜急性损伤可能与其增加胃粘膜血流量有关,与血EGF和CGRP无关。     

人ZP3基因的RT-PCR cDNA克隆

目的：研究人ZP3基因的结构及构建人ZP3基因原核表达系统。方法：从人卵巢组织中分离出mRNA并以此作为模版,通过RT—PCR扩增出人ZP3基因cDNA片段,然后将其克隆在pUC18质粒上,并对克隆片段进行序列分析。结果：共克隆到ZP3-A（1300bp）、ZP3-B（1180bp）、ZP3-C（1200bp）和ZP3-D（1080bp）4种不同长度的人ZP3基因cDNA片段,对其中最长的ZP3-A片段的测序结果表明,它包含了人ZP3基因阅读框内的全部序列,与NCBI Sequence Viewer中公布的人ZP3 mRNA序列（NM-007155）相比较,在1275bp长的编码区内只有一个碱基不同,两者同源性达到99．92％。结论：本研究克隆到的ZP3-A cDNA片段确是人ZP3基因无疑。     

特发性少精子症和无精子症与Pygo2基因蛋白编码区SNPs的相关性

男性不育常伴随精子数量减少。Pygo2基因在染色质重塑的伸长精细胞中表达, 其功能受损会导致精子形成阻滞和精子生成减少而引发不育。文章旨在检测引起人特发性少精子症和无精子症的Pygo2基因突变。从77例正常生育力男性和195例特发性少精子症和无精子症患者静脉血提取DNA, 采用聚合酶链式反应-测序方法对Pygo2基因3个蛋白质编码区进行测序对比, 非同义单核苷酸多态性(Single nucleotide polymorphisms, SNPs)位点分别用SIFT、Polyphen-2和 Mutation Taster软件进行诱发蛋白质结构和表型改变的检测和分析。结果表明, 195例患者中, 178例(30例轻度或中度少精子症, 57例重度少精子症和91例无精子症)基因序列分析报告完好, 无精子症中3例患者分别在2个位点(rs61758740, rs141722381)发生了非同义突变SNPs, 重度少精子症中1例患者在位点rs61758741发生了非同义突变, 3个突变位点在SNPs基因数据库都已有报道, 轻度或中度少精子症患者以及正常生育力男性中不存在SNPs。rs61758740可使PYGO2蛋白第141位蛋氨酸(M)变为异亮氨酸(I), rs61758741使PYGO2蛋白第261位碱性赖氨酸(K)变为酸性谷氨酸(E), rs141722381使PYGO2蛋白第240位亲水侧链天冬酰胺(N)变为疏水侧链异亮氨酸(I)。软件分析表明, 在所发现的3个SNP非同义突变位点中, rs141722381引起的单个氨基酸改变会导致PYGO2蛋白空间结构破坏和诱发相关疾病。因此, Pygo2基因蛋白质编码序列区SNPs可能是特发性少精子症和无精子症的诱发因素之一, 导致男性不育。     

A comparison of Bonus and Quota mating systems for utilising the sex-determining region Y gene in terminal sire beef cattle breeding

The use of the sex-determining region Y gene in terminal sire beef cattle breeding was investigated, assuming that fertile transgenic bulls carrying this gene on an autosome can be created. The benefit of these transgenic bulls arises from having an increased proportion of calves with a male phenotype. Two mating strategies utilising the transgenic bulls were devised and compared; the Quota scheme whereby a quota of normal bulls is used alongside the transgenic bulls in a breeding nucleus, and the Bonus scheme in which a phenotypic bonus is assigned to transgenic bulls indicating the added value of their offspring. Bonus and Quota breeding schemes were comparable, in terms of the value of offspring of the bulls, for the first 6 years of selection, after which time the Quota breeding scheme was superior. For time horizons less than 10 years, and large assumed phenotypic superiorities of male calves, breeding schemes with transgenic bulls were superior to traditional breeding schemes without transgenic bulls. If the time horizon was longer, or if the assumed superiority of male calves was small, then traditional breeding schemes were generally superior to those utilising transgenic bulls. Scenarios were observed, however, where transgenic bulls were always superior to normal bulls, in terms of their value as sires. Equations were derived to predict genetic gain and the equilibrium genetic lag between normal and transgenic bulls in Quota breeding schemes.     

shRNA抑制宫颈癌Hela细胞株HPV18 E6、E7基因表达的研究

应用短发夹RNA(Short hairpin RNA,shRNA)表达载体抑制宫颈癌Hela细胞株HPV18 E6、E7基因的表达。应用已鉴定的shRNA表达载体pHPV1、pHPV2转染Hela细胞,G418筛选阳性细胞,建立稳定转染细胞株;倒置荧光显微镜检测转染情况;提取细胞内总RNA,RT-PCR方法检测HPV18 E6、E7 mRNA;WesternBlot检测HPV18 E6、E7蛋白表达的变化;采用灰度分析软件对PCR扩增条带与蛋白质条带进行灰度分析。pHPV1实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的31%、38%,E6、E7蛋白分别为阴性对照组的37%、31%;pHPV2实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的54%、77%,E6、E7蛋白分别为阴性对照组的52%、83%。pHPV1、pHPV2表达载体能抑制Hela细胞HPV18 E6、E7的表达,针对外显子区434-452的pHPV1抑制作用更强。     

应用基因芯片筛选鼻咽癌信号转导相关基因

目的：建立具有组织特异性的鼻咽癌基因表达谱,筛选鼻咽癌中信号转导相关基因。方法：采用深圳微芯公司基于玻片的包含8046个人类基因的基因芯片,检测7例鼻咽癌组织及1例鼻咽炎组织,初步获得鼻咽癌异常表达基因;结合GO分类从异常表达的基因中筛选信号转导相关基因,以Biocarta信号通路数据库查询筛选基因相关转导信号通路信息。结果：在鼻咽癌组织独得1241个异常用表达基因,其中高表达基因871个,低表达基因343个。发现28个差异表达基因与细胞的信号转导相关,其中表达上调的21个,表达下调的7个。结论：成功建立了具有组织特异性的鼻咽癌基因表达谱,初步获得了鼻咽癌信号转导相关基因。