龚氏生物反应堆_创建与应用

龚氏重组成的生物反应堆(以下简称为GRB)是用转交法将克隆成的供体DNA转入受体创建而成的.基因表达表明这一生物反应堆能耐高剂量盐(NaCl3.2M)、抗生素(四环素Te100μg/ml和三甲氧苄二氨嘧啶Trim 50μg/ml)以及毒素(铃兰氨酸Azet 100μg/ml).这一生物反应堆的后代对盐、抗生素和毒素保持同样高的耐力.这表明这一生物反应堆的耐力能代代相传.在波士顿、旧金山.深圳和青岛的海水加Te(100μg/ml)的条件下,它能生长;在深圳或青岛海水加Te(200μg/ml),矿物质和碳源的条件下,也能迅速增殖(干物质1.6mg/ml/24hrs);蛋白质产量为0.59mg/ml,胞外多糖为0.26mg/ml.     

斜纹夜蛾核多角体病毒egt基因的克隆和部分序列分析

用ＡｃＭＮＰＶｅｇｔ基因中的保守区部分片段为探针,通过Ｓｏｕｔｈｅｒｎ杂交确定ＳｌＭＮ－ＰＶｅｇｔ基因的位置在ＰｓｔⅠ４９７０ｂｐ和ＸｂａⅠ２６００ｂｐ的片段上．采用双链ＤＮＡ序列分析方法测序,在２６００ｂｐ片段上的ＥｃｏＲⅠ位点两侧得到５９４ｂｐＤＮＡ碱基序列,用计算机ＤＮＡＳＩＳ和ＰＲＯＳＩＳ软件,将５９４ｂｐＤＮＡ碱基序列所推测的氨基酸序列与ＡｃＭＮＰＶ和ＳｌｉＭＮＰＶ的ｅｇｔ基因氨基酸序列进行同源比较,其同源性分别为４５％和８６．５％．     

Transgenesis in fish

Gene transfer into fish embryo is being performed in several species (trout, salmon, carps, tilapia, medaka, goldfish, zebrafish, loach, catfish, etc.). In most cases, pronuclei are not visible and microinjection must be done into the cytoplasm of early embryos. Several million copies of the gene are generally injected. In medaka, transgenesis was attempted by injection of the foreign gene into the nucleus of oocyte. Several reports indicate that the injected DNA was rapidly replicated in the early phase of embryo development, regardless of the origin and the sequence of the foreign DNA. The survival of the injected embryos was reasonably good and a large number reached maturity. The proportion of transgenic animals ranged from 1 to 50% or more, according to species and to experimentators. The reasons for this discrepancy have not been elucidated. In all species, the transgenic animals were mosaic. The copy number of the foreign DNA was different in the various tissues of an animal and a proportion lower than 50% of F1 offsprings received the gene from their parents. This suggests that the foreign DNA was integrated into the fish genome at the two cells stage or later. An examination of the integrated DNA in different cell types of an animal revealed that integration occurred mainly during early development. The transgene was found essentially unrearranged in the fish genome of the founders and offsprings. The transgenes were therefore stably transmitted to progeny in a Mendelian fashion. Southern blot analysis revealed the presence of possible junction fragments and also of minor bands which may result from a rearrangement of the injected DNA. In all species, the integrated DNA appeared mainly as random end-to-end concatemers. In adult trout blood cells, a small proportion of the foreign DNA was maintained in the form of non-integrated concatemers, as judged by the existence of end fragments. The transgenes were generally only poorly expressed. The majority of the injected gene constructs contained essentially mammalian or higher vertebrates sequences. The comparison of the expression efficiency of these constructs in transfected fish and mammalian cells indicates that some of the mammalian DNA sequences are most efficiently understood by the fish cell machinery. Chloramphenicol acetyl transferase gene under the control of promoters from Rous sarcoma virus, and human cytomegalovirus, was expressed in several tissues of transgenic fish. Chicken -crystallin gene was expressed in several tissues of transgenic fish. Rainbow trout growth hormone cDNA driven by the Rous sarcoma virus promoter was expressed in transgenic carps leading to a faster growth of these animals. The antifreeze protein gene from flounder was expressed in transgenic salmon. These data indicate that transgenesis in fish is relatively easy but that fish gene sequences must be preferably used to obtain a good expression of the transgenes. Fish is a good biological model, specially for developmental studies and it is an increasing part of human food. For these reasons, transgenesis in fish is most likely to be more and more practised in the coming years.     

未知二粒子纠缠态及其正交态的概率克隆

提出一个实现二粒子纠缠态及其正交态的概率克隆方案.此方案分为两个步骤,第一步通过一个四粒子簇态作为量子信道实现未知二粒子纠缠态的量子隐形传输,第二步在态的制备者的帮助下实现二粒子纠缠态及其正交态的概率克隆.     

β-1,4-葡聚糖内切酶基因的克隆及在大肠杆菌中的表达

利用PCR方法从一株短小芽孢杆菌H9克隆了编码葡聚糖内切酶的基因（该序列已经被已收录于GeneBank,登录号为EF620915）,序列分析表明该基因读码框为1980bp,编码659个氨基酸。预测的酶由两个不连续的结构域组成,其一为N-端催化结构域,由糖基水解酶家族9组成,第二个结构域为C-端底物结合结构域,由碳水化合物绑定结构域家族3组成。将基因构建在大肠杆菌表达载体pET20b中得到重组质粒pET20b-EglA,将重组载体转化至大肠菌株BL21(DE3)菌株,基因在大肠杆菌中得到了良好的分泌表达, SDS-电泳图谱表明酶的分子大小约为73KD。     

Comparative pluripotency analysis of mouse embryonic stem cells derived from wild-type and infertile hermaphrodite somatic cell nuclear transfer blastocysts

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer （SCNT）, holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells （hESCs） have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmis- sion, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi- or pluripotency has not yet been evaluated. In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer- ESC （ntESC） lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results in- dicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.     

青稞脱水素基因dhn4的克隆与原核表达

用RT-PCR的方法从重要的高原作物青稞(Hordeum vulgare L.vat.nudum Hook.f.)中克隆到了脱水素基因dhn4,其编码区长为678 bp,所编码的蛋白质DHN4为YSK2型脱水素.经预测,该蛋白二级结构易形成α-螺旋,并无固定的三级结构.将dhn4基因cDNA编码区定向克隆到载体PEW30-c上,获得了重组质粒PET-dhn4,该重组质粒再转化到大肠杆菌BL21茵株,37℃经1.5 mmol/L IPTG诱导获得了大量分子量约为35 kD的融合蛋白.然后用亲和层析的方法对该蛋白进行纯化,得到了纯化的脱水素DHN4蛋白.     

酿酒酵母蔗糖酶基因的克隆、异源表达及重组酶学性质研究

以酿酒酵母(Saccharomyces cerevisiae)基因组DNA为模板,通过PCR扩增得到蔗糖酶基因(suc2),并将其克隆到表达载体pSE380中,将得到的重组质粒pSE-suc2转化进E.coli BL21中,利用镍金属螯合层析方法分析测定其酶学性质。重组菌株的SDS-PAGE结果显示重组蔗糖酶基因(suc 2)有60kDa目的蛋白出现,纯化的SDS-PAGE分析得到均一的蛋白条带;重组蔗糖酶的Km值为47.73mmol/L,最大反应速率为79.59mg还原糖mg-1蛋白min-1,最适温度为42℃,最适pH值为5.5,Zn2 、Cu2 对重组蔗糖酶酶活有较强的抑制作用,Ba2 、Mg2 、Mn2 对重组蔗糖酶酶活稍有激活作用。     

Differential expression analysis and regulatory network reconstruction for genes associated with muscle growth and adipose deposition in obese and lean pigs

During the growth and development of skeletal muscle cells and adipose cells, the regulatory mechanism of micro-effect polygenes determines porcine meat quality, carcass characteristics and other relative quantitative traits. Obese and lean type pig breeds show obvious differences in muscle growth and adipose deposition; however, the molecular mechanism underlying this phenotypic variation remains unknown. We used pathway-focused oligo microarray studies to examine the expression changes of 140 genes associated with muscle growth and adipose deposition in longissimus dorsi muscle at six growth stages （birth, 1, 2, 3, 4 and 5 months） of Landrace （a leaner, Western breed） and Taihu pigs （a fatty, indigenous, Chinese breed）. Variance analysis （ANOVA） revealed that differences in the expression of 18 genes in Landrace pigs and three genes in Taihu pigs were very significant （FDR-adjusted permutation, P 〈 0.01） and differences for 22 genes in Landrace pigs and seven genes in Taihu pigs were significant （FDR-adjusted permutation, P 〈 0.05） among six growth stages. Clustering analysis revealed a high level of significance （FDR-adjusted, P 〈 0.01） for four gene expression patterns, in which genes that strongly up-regulated were mainly associated with the positive regulation of myofiber formation and fatty acid biogenesis and genes that strongly down-regulated were mainly associated with the inhibition of cell proliferation and positive regulation of fatty acid β-oxidation. Based on a dynamic Bayesian network （DBN） model, gene regulatory networks （GRNs） were reconstructed from time-series data for each pig breed. These two GRNs initially revealed the distinct differences in physiological and biochemical aspects of muscle growth and adipose deposition between the two pig breeds; from these results, some potential key genes could be identified. Quantitative real-time RT-PCR （QRT-PCR） was used to verify the microarray data for five modulated genes, and a good correlation between     

Conditional and unconditional mapping of quantitative trait loci underlying plant height and tiller number in rice (Oryza sativa L.) grown at two nitrogen levels

In this study,we used 127 double haploid (DH) lines to analyze agricultural traits of rice.The DH lines,derived from a ZYQ8 (indica)/JX17 (japonica) cross by anther culture,contained 160 RFLP and 83 SSR markers.Unconditional and conditional quantitative trait loci (QTL) mapping was conducted to analyze plant height (PH) and tillers per plant (TP) at five growth stages that were grown at two nitrogen levels.Fourteen PH and 13 TP unconditional QTL were identified in the different growth stages,including 19 QTL from high-nitrogen (HN) and 14 QTL from low-nitrogen (LN) conditions.The conditional QTL for 14 genomic regions under LN/HN con-ditions showed that there was a significant effect on PH and TP across the different stages.Only one conditional QTL,ph2-3,was unable to be detected in unconditional mapping.More QTL were detected in the first four rice growth stages than in the final stage.Further-more,a line from the DH mapping population,DH78,was identified in extreme phenotypes of PH and TP that exhibited dwarfism and less-tiller (dft) characters.The gene dftl was mapped to chromosome 2 using a backcrossed population of DH78/JX17 through a map-based cloning strategy.The location of dftl coincided with the mapping region of the small-LOD peak,QTL ph2 and tp2,which were identified in plants grown in low-nitrogen conditions.Further backcrossing and fine-mapping successfully delimited the dftl locus to a 91 kb region.