利用实时荧光定量比较Ct法检测转基因小鼠外源基因拷贝数

目的：在建立转基因小鼠模型时,外源基因拷贝数是影响其表达水平和遗传稳定性的重要因素之一。外源基因拷贝数的精确测定,是建立转基因动物模型的重要环节。方法：合成cagA基因和内参基因GAPDH的引物,用标准曲线法测得cagA和GAPDH基因的扩增效率分别为97．6％和98．6％;将128拷贝阴性小鼠基因组和128拷贝c0鲥打靶质粒的混合物作为参照样品,取6只来自同一母本的F2阳性小鼠的128拷贝基因组作为待测样品;选取GAPDH作为内源参照基因,用比较Ct法对待测样品进行定量。结果：经计算,6只待测小鼠的cagA基因拷贝数平均值为8。结论：利用实时荧光定量PCR仪,呆用改良后的比较Ct法对转基因小鼠的外源基因拷贝数进行了精确测定。     

Large deletion of androsterone UDP-glucuronosyltransferase gene in the inherited deficient strain of Wistar rats

LA Wistar rats have a deficiency of androsterone UDP-glucuronosyltransferase (UDPGT) and are present in Wistar rat colonies around the world. In order to clarify the molecular mechanism of the deficiency, androsterone UDPGT cDNA clone, pGT2 was isolated from rat liver cDNA library and was digested with restriction enzymes to afford three probes for Northern and Southern blot analyses in HA (normal), heterozygous LA and LA Wistar rats. In Northern blot analysis, androsterone UDPGT mRNA was totally absent in LA Wistar rat liver. Southern blot analysis suggested a large deletion of androsterone UDPGT gene in the rats. Genomic DNA amplifications with synthetic primers which have nucleotide sequences corresponding to the 5′-region of androsterone UDPGT cDNA, suggested that androsterone UDPGT gene has exon 1 with a length of some 700 bp and that this exon is deleted in LA Wistar rats. Based on these lines of evidence, it is concluded that the large portion of androsterone UDPGT gene is deleted in LA Wistar rats, which results in the absence of androsterone UDPGT mRNA and consequently the corresponding enzyme protein.     

Functional expression of the Flp recombinase in Mycobacterium bovis BCG

Mycobacteria contain a large number of redundant genes whose functions are difficult to analyze in mutants because there are only two efficient antibiotic resistance genes available for allelic exchange experiments. Sequence-specific recombinbases such as the Flp recombinase can be used to excise resistance markers. Expression of the flp(e) gene from Saccharomyces cerevisiae is functional for this purpose in fast-growing Mycobacterium smegmatis but not in slow-growing mycobacteria such as M. bovis BCG or M. tuberculosis. We synthesized the flp(m) gene by adapting the codon usage to that preferred by M. tuberculosis. This increased the G+C content from 38% to 61%. Using the synthetic flp(m) gene, the frequency of removal of FRT-hyg-FRT cassette from the chromosome by the Flp recombinase was increased by more than 100-fold in M. smegmatis. In addition, 40% of all clones of M. bovis BCG had lost the hyg resistance cassette after transient expression of the flp(m) gene. Sequencing of the chromosomal DNA showed that excision of the FRT-hyg-FRT cassette by Flp was specific. These results show that the flp(m) encoded Flp recombinase is not only an improved genetic tool for M. smegmatis, but can also be used in slow growing mycobacteria such as M. tuberculosis for constructing unmarked mutations. Other more sophisticated applications in mycobacterial genetics would also profit from the improved Flp/FRT system.     

Molecular cloning,characterization and expression analysis of ATG1 in the silkworm, Bombyx mori

Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process.     

The Jumonji gene family in Crassostrea gigas suggests evolutionary conservation of Jmj-C histone demethylases orthologues in the oyster gametogenesis and development

Jumonji (Jmj) proteins are histone demethylases, which control the identity of stem cells. Jmj genes were characterized from plants to mammals where they have been implicated in the epigenetic regulation of development. Despite the Pacific oyster Crassostrea gigas representing one of the most important aquaculture resources worldwide, the molecular mechanisms governing the embryogenesis and reproduction of this lophotrochozoan species remain poorly understood. However, annotations in the C. gigas EST library suggested the presence of putative Jumonji genes, raising the question of the conservation of this family of histone demethylases in the oyster.     

The evolution and functional divergence of the beta-carotene oxygenase gene family in teleost fish—Exemplified by Atlantic salmon

In mammals, two carotenoid cleaving oxygenases are known; beta-carotene 15,15′-monooxygenase (BCMO1) and beta-carotene 9′,10′-oxygenase (BCO2). BCMO1 is a key enzyme in vitamin A synthesis by symmetrically cleaving beta-carotene into 2 molecules of all-trans-retinal, while BCO2 is responsible for asymmetric cleavage of a broader range of carotenoids. Here, we show that the Atlantic salmon beta-carotene oxygenase (bco) gene family contains 5 members, three bco2 and two bcmo1 paralogs. Using public sequence databases, multiple bco genes were also found in several additional teleost species. Phylogenetic analysis indicates that bco2a and bco2b originate from the teleost fish specific genome duplication (FSGD or 3R), while the third and more distant paralog, bco2 like, might stem from a prior duplication event in the teleost lineage. The two bcmo1 paralogs (bcmo1 and bcmo1 like) appear to be the result of an ancient duplication event that took place before the divergence of ray-finned (Actinopterygii) and lobe-finned fish (Sarcopterygii), with subsequent nonfunctionalization and loss of one Sarcopterygii paralog. Gene expression analysis of the bcmo1 and bco2 paralogs in Atlantic salmon reveals regulatory divergence with tissue specific expression profiles, suggesting that the beta-carotene oxygenase subtypes have evolved functional divergences. We suggest that teleost fish have evolved and maintained an extended repertoire of beta-carotene oxygenases compared to the investigated Sarcopterygii species, and hypothesize that the main driver behind this functional divergence is the exposure to a diverse set of carotenoids in the aquatic environment.     

Identification,expression and bioactivity of hexokinase in amphioxus: Insights into evolution of vertebrate hexokinase genes

Hexokinase family includes hexokinases I, II, III and IV, that catalyze the phosphorylation of glucose to produce glucose 6-phosphate. Hexokinase IV, also known as glucokinase, is only half size of the other types of hexokinases that contain two hexokinase domains. Despite the enormous progress in the study of hexokinases, the evolutionary relationship between glucokinase and other hexokinases is still uncertain, and the molecular processes leading to the emergence of hexokinases in vertebrates remain controversial. Here we clearly demonstrated the presence of a single hexokinase-like gene in the amphioxus Branchiostoma japonicum, Bjhk, which shows a tissue-specific expression pattern, with the most abundant expression in the hepatic caecum, testis and ovary. The phylogenetic and synteny analyses both reveal that BjHK is the archetype of vertebrate hexokinases IV, i.e. glucokinases. We also found for the first time that recombinant BjHK showed functional enzyme activity resembling vertebrate hexokinases I, II, III and IV. In addition, a native glucokinase activity was detected in the hepatic caecum. Finally, glucokinase activity in the hepatic caecum was markedly reduced by fasting, whereas it was considerably increased by feeding. Altogether, these suggest that Bjhk represents the archetype of glucokinases, from which vertebrate hexokinase gene family was evolved by gene duplication, and that the hepatic caecum plays a role in the control of glucose homeostasis in amphioxus, in favor of the notion that the hepatic caecum is a tissue homologous to liver.     

Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants - a review

The increasing effect of non-degradable plastic wastes is a growing concern. Polyhydroxyalkanoates (PHAs), macromolecule-polyesters naturally produced by many species of microorganisms, are being considered as a replacement for conventional plastics. Unlike petroleum-derived plastics that take several decades to degrade, PHAs can be completely bio-degraded within a year by a variety of microorganisms. This biodegradation results in carbon dioxide and water, which return to the environment. Attempts based on various methods have been undertaken for mass production of PHAs. Promising strategies involve genetic engineering of microorganisms and plants to introduce production pathways. This challenge requires the expression of several genes along with optimization of PHA synthesis in the host. Although excellent progress has been made in recombinant hosts, the barriers to obtaining high quantities of PHA at low cost still remain to be solved. The commercially viable production of PHA in crops, however, appears to be a realistic goal for the future.     

The polymorphism in the promoter region of metallothionein 1 is associated with heat tolerance of scallop Argopecten irradians

Metallothioneins (MTs), a superfamily of cysteine-rich proteins, perform multiple functions, such as maintaining homeostasis of essential metals, detoxification of toxic metals and scavenging of oxyradicals. In this study, the promoter region of a metallothionein (MT) gene from Bay scallop Argopecten irradians (designed as AiMT1) was cloned by the technique of genomic DNA walking, and the polymorphisms in this region were screened to find their association with susceptibility or tolerance to high temperature stress. One insert–deletion (ins–del) polymorphism and sixteen single nucleotide polymorphisms (SNPs) were identified in the amplified promoter region. Two SNPs, − 375 T–C and − 337 A–C, were selected to analyze their distribution in the two Bay scallop populations collected from southern and northern China coast, which were identified as heat resistant and heat susceptible stocks, respectively. There were three genotypes, T/T, T/C and C/C, at locus − 375, and their frequencies were 25%, 61.1% and 13.9% in the heat susceptible stock, while 34.2%, 42.1% and 23.7% in the resistant stock, respectively. There was no significant difference in the frequency distribution of different genotypes between the two stocks (P > 0.05). In contrast, at locus − 337, three genotypes A/A, A/C and C/C were revealed with the frequencies of 11.6%, 34.9% and 53.5% in the heat susceptible stock, while 45.7%, 32.6% and 21.7% in the heat resistant stock, respectively. The frequency of C/C genotype in the heat susceptible stock was significantly higher (P < 0.01) than that in the heat resistant stock, while the frequency of A/A in the heat resistant stock was significantly higher (P < 0.01) than that in the heat susceptible stock. Furthermore, the expression of AiMT1 mRNA in scallops with C/C genotype was significantly higher than that with A/A genotype (P < 0.05) after an acute heat treatment at 28 °C for 120 min. These results implied that the polymorphism at locus − 337 of AiMT1 was associated with the susceptibility/tolerance of scallops to heat stress, and the − 337 A/A genotype could be a potential marker available in future selection of Bay scallop with heat tolerance.     

Genomic structure, polymorphism and expression analysis of the growth hormone (GH) gene in female and male Half-smooth tongue sole (Cynoglossus semilaevis)

Growth hormone (GH) is a polypeptide which is an important regulator of development and somatic growth in teleosts, and may be associated with the mechanisms which drive sexual growth dimorphism in the Half-smooth tongue sole (Cynoglossus semilaevis). In this study, the full length gh cDNA was cloned from C. semilaevis by homology cloning and the rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). The full-length gh cDNA is 826 bp and contains an open reading frame (ORF) of 603 bp encoding a protein of 200 amino acids (AA). The precursor of gh consists of a 17 amino-acid signal peptide followed by a 183 amino-acid mature polypeptide. GH gene sequences obtained from female and male adults consist of 3428 bp and 3371 bp, respectively, each of which includes six exons and five introns, and the difference in the GH gene size was mainly caused by the microsatellites. When 14 tissues from females, normal males and extra-large male adults were analyzed for sex-specific tissue expression, the gh mRNA was found to be predominantly expressed in the pituitary, and the expression levels in females were 3.6 times as much as those in normal males, while the mRNA expression in extra-large males was 1.7 times as much as those in normal males. Sex differences in gh mRNA expression during development were also examined by using a full-sib family of C. semilaevis, and the gh mRNA was detected at all of the 12 time points sampled from 10 to 380 days-old. A significant increase in gh mRNA was detected starting in 80 day old fish and was then followed by a drop to very low levels starting at 230 day old fish. Differential expression indicated that the gh expression level in females was significantly higher than males (P < 0.01) at all of the stages except for 10 days-old. Two microsatellite loci were identified in the second intron of the GH gene. Using these two polymorphic markers to genotype 224 individuals, there was no significant difference between the females and males in the Bohai Sea, the Yellow Sea and the hatchery samples.     

Cloning,expression and cellular localization of the Doublesex gene in the water flea, Daphnia carinata, during different developmental stages

In this study, one of Doublesex genes from the common freshwater cladoceran Daphnia carinata, designated DapcaDsx1, was cloned using primers based on homologous sequences and rapid amplification of cDNA ends (RACE). qPCR was employed to quantify differences in DapcaDsx1 expression between the different sexual phases, with expression levels being higher in sexual females. The role of DapcaDsx1 in the reproductive transformation was further investigated in parthenogenetic-phase females and sexual-phase females using whole-mount in situ hybridization. This cellular localization study showed specific expression of DapcaDsx1 in the thoracic segments, second antenna and part of the ventral carapace. Higher expression levels were exhibited in sexual females compared to parthenogenetic females. This suggests that the DapcaDsx1 gene plays significant roles in switching modes of reproduction and during sexual differentiation.