Molecular cloning and functional characterization of MdPIN1 in apple

Auxin has been identified to play critical roles in regulating plant growth and development. The polar transport of auxin is regulated by auxin transporters. In the present study, an auxin efflux carrier gene MdPIN1 was cloned from Malus×domestic, Royal Gala, and introduced into wild-type Arabidopsis thaliana(Col-0). The transgenic plants exhibited the phenotype of inhibition of primary root(PR) elongation and increased lateral root(LR) number in compared with Col-0. Overexpression of Md PIN1 affected auxin transport, and enhanced phototropic responses and geotropism reaction, whereas had no significant difference in the auxin biosynthesis. These findings suggest that the Md PIN1 gene plays a vital role in auxin transport and root development.     

拟南芥xtc1突变体的图位克隆分析

【目的】分析拟南芥xtc1突变体茎部表皮蜡的组分和含量,并鉴定导致xtc1突变体表型的基因。【方法】通过气相色谱法分析拟南芥xtc1突变体与Ler野生型茎部表皮蜡的成分;利用图位克隆确定突变基因位点,通过在拟南芥xtc1突变体中过量表达FATB基因,验证突变位点与FATB基因的关系。【结果】拟南芥xtc1突变体茎部表皮蜡总量约为Ler野生型的1/3,且各组分含量均明显减少;将突变基因定位在第1染色体顶端物理距离为80kb的2个标记T27G7-3和F22O13-1之间,该区域含有21个基因。T-DNA插入突变体观察及测序分析表明,xtc1突变体在At1g08510(FATB)基因的第1个外显子上产生14个碱基的缺失,导致翻译提前终止;在xtc1突变体中过量表达FATB基因可恢复xtc1突变体的正常表型。【结论】拟南芥xtc1突变体茎部表皮蜡含量减少,且突变基因为FATB基因。     

哺乳动物克隆技术研究进展

哺乳动物克隆是目前生物技术领域研究的热点之一 ,具有重大的科学意义和应用价值。文章着重介绍了哺乳动物克隆细胞核移植方法和进展 ,以及体细胞核移植的应用前景     

A mutation of the circadian system in golden hamsters

A mutation has been found that dramatically shortens the period of the circadian locomotor rhythm of golden hamsters. The pattern of inheritance of this mutation suggests that it occurred at a single, autosomal locus (tau). Wild-type animals have rhythms with free-running periods averaging about 24 hours; animals heterozygous for the mutation have periods of about 22 hours, whereas homozygous animals have rhythms with periods close to 20 hours. Animals that carry the mutant alleles exhibit abnormal entrainment to 24-hour light:dark cycles or are unable to entrain.     

Positional cloning of the human quantitative trait locus underlying taste sensitivity to phenylthiocarbamide

The ability to taste the substance phenylthiocarbamide (PTC) has been widely used for genetic and anthropological studies, but genetic studies have produced conflicting results and demonstrated complex inheritance for this trait. We have identified a small region on chromosome 7q that shows strong linkage disequilibrium between single-nucleotide polymorphism (SNP) markers and PTC taste sensitivity in unrelated subjects. This region contains a single gene that encodes a member of the TAS2R bitter taste receptor family. We identified three coding SNPs giving rise to five haplotypes in this gene worldwide. These haplotypes completely explain the bimodal distribution of PTC taste sensitivity, thus accounting for the inheritance of the classically defined taste insensitivity and for 55 to 85% of the variance in PTC sensitivity. Distinct phenotypes were associated with specific haplotypes, which demonstrates that this gene has a direct influence on PTC taste sensitivity and that sequence variants at different sites interact with each other within the encoded gene product.     

哺乳动物体细胞克隆的研究与应用

通过查阅研究1997年以来国内外对哺乳动物体细胞克隆技术的研究文献,综述了哺乳动物体细胞克隆技术的发展历史、研究现状、影响因素及存在问题,并对其应用前景进行了探讨。     

Norwalk virus genome cloning and characterization

Major epidemic outbreaks of acute gastroenteritis result from infections with Norwalk or Norwalk-like viruses. Virus purified from stool specimens of volunteers experimentally infected with Norwalk virus was used to construct recombinant complementary DNA (cDNA) and derive clones representing most of the viral genome. The specificity of the clones was shown by their hybridization with post- (but not pre-) infection stool samples from volunteers infected with Norwalk virus and with purified Norwalk virus. A correlation was observed between the appearance of hybridization signals in stool samples and clinical symptoms of acute gastroenteritis in volunteers. Hybridization assays between overlapping clones, restriction enzyme analyses, and partial nucleotide sequence information of the clones indicated that Norwalk virus contains a single-stranded RNA genome of positive sense, with a polyadenylated tail at the 3' end and a size of at least 7.5 kilobases. A consensus amino acid sequence motif typical of viral RNA-dependent RNA polymerases was identified in one of the Norwalk virus clones. The availability of Norwalk-specific cDNA and the new sequence information of the viral genome should permit the development of sensitive diagnostic assays and studies of the molecular biology of the virus.     

Porcine LEM domain-containing 3: Molecular cloning,functional characterization,and polymorphism associated with ear size

Ear size exhibits remarkable diversity in pig breeds. LEM domain-containing 3(LEMD3) on chromosome 5 is considered as an important candidate for porcine ear size. This is the first study on cloning and characterization of LEMD3 c DNA. The complete c DNA contains 4 843 bp, including a 2 736-bp open reading frame(ORF), a 37-bp 5′-untranslated region(UTR) and a 2 070-bp 3′-UTR. The complete LEMD3 gene is 126 241-bp and contains 13 exons and 12 introns. The ORF encodes a deduced LEMD3 protein of 911 amino acids, which shares 82–94% nucleic acid and 51–96% amino acid identity with other species. A phylogenetic tree constructed based on the amino acid sequences revealed that the porcine LEMD3 protein was closely related with cattle LEMD3. Resequencing of the ORF and promoter of LEMD3 from Minzhu pig and Large White revealed three single nucleotide polymorphisms(SNPs): L964CA in the complete coding region, L4625AG in the 3′ UTR, and L-394TC in the promoter region. Genome-wide association study(GWAS) revealed that all of SNPs were shown significant association with ear size in Large White×Minzhu pig intercross population. With conditional GWAS, –log10(P-value) decreased by more than 80% when each of three SNPs was included as a fixed effect. These results suggested direct involvement of LEMD3 or close linkage to the causative mutation for ear size. The findings of this study might form the basis for understanding the genetic mechanism of ear size variation in pigs and provide potential molecular markers for screening ear size diversity in pig breeds.     

Regulation of mammalian circadian behavior by non-rod, non-cone, ocular photoreceptors

Circadian rhythms of mammals are entrained by light to follow the daily solar cycle (photoentrainment). To determine whether retinal rods and cones are required for this response, the effects of light on the regulation of circadian wheel-running behavior were examined in mice lacking these photoreceptors. Mice without cones (cl) or without both rods and cones (rdta/cl) showed unattenuated phase-shifting responses to light. Removal of the eyes abolishes this behavior. Thus, neither rods nor cones are required for photoentrainment, and the murine eye contains additional photoreceptors that regulate the circadian clock.     

两个类ADP-葡萄糖转运蛋白基因的克隆和特征

搜索数据库获得了2个新的类似ADP-葡萄糖转运蛋白(BT)基因的序列,通过PCR直接克隆了这2个基因。这2个基因都编码406个氨基酸组成的分子量为43 647Da的蛋白。BT2 A在蛋白氮端有一段59个氨基酸残基组成的叶绿体信号肽,而BT2 B的叶绿体信号肽长度为58个氨基酸。BT2 A在信号肽后的是一段71个氨基酸长可变区,碳端是276个氨基酸长的功能域;BT2 B可变区由72个氨基酸组成,碳端也是276氨基酸长的功能域。半定量PCR表明,BT2 A为组成型表达,而BT2 B则在胚乳发育的中晚期表达。BT基因进化树分析表明,BT2 A和BT2 B基因是在禾谷类作物分化后由基因倍增产生的。     

Molecular cloning and functional expression of glutamate receptor subunit genes

Three closely related genes, GluR1, GluR2, and GluR3, encode receptor subunits for the excitatory neurotransmitter glutamate. The proteins encoded by the individual genes form homomeric ion channels in Xenopus oocytes that are sensitive to glutamatergic agonists such as kainate and quisqualate but not to N-methyl-D-aspartate, indicating that binding sites for kainate and quisqualate exist on single receptor polypeptides. In addition, kainate-evoked conductances are potentiated in oocytes expressing two or more of the cloned receptor subunits. Electrophysiological responses obtained with certain subunit combinations show agonist profiles and current-voltage relations that are similar to those obtained in vivo. Finally, in situ hybridization histochemistry reveals that these genes are transcribed in shared neuroanatomical loci. Thus, as with gamma-aminobutyric acid, glycine, and nicotinic acetylcholine receptors, native kainate-quisqualate-sensitive glutamate receptors form a family of heteromeric proteins.     

猪EGR2基因外显子2变异位点分析

早期生长反应因子2(EGR2,Krox20)能够参与脂肪代谢并具有可以结合靶基因启动子富含GC序列的3个锌指的转录极早期转录调控因子。EGR2能够调控CCAAT增强子结合蛋白Beta(C/EBPβ)、类Krupple因子5(KLF5)、过氧化物酶体增殖物激活受体Gamma(PPARγ)的表达来参与对脂肪代谢的影响。对EGR2基因外显子2进行克隆测序,获得1247bp序列,并利用PCR-SSCP方法对其进行分子扫描,寻找多态位点,分析不同基因型在不同猪种间的分布规律。χ2独立性检验表明,野猪、民猪、大白猪、杜洛克、长白猪间不同基因型的分布存在着极其显著的差异(P<0.01)。     

哺乳动物MT基因的进化选择与功能分歧

利用生物信息学的方法,使用PAML和DIVERGE等软件对金属硫蛋白进行研究,发现哺乳动物MT基因主要受到纯化选择及中性选择作用,而且其在进化历史中曾经受到过正选择作用。同时利用PAML软件检测到了4个受到正选择的氨基酸位点,利用DIVERGE软件检测到5个Ⅰ型功能分歧位点和2个Ⅱ型功能分歧位点。这些正选择位点和功能分歧位点为进一步研究金属硫蛋白的结构与功能提供了参考和依据。     

Molecular cloning and functional characterization of apple U-box E3 ubiquitin ligase gene MdPUB29 reveals its involvement in salt tolerance

An E3 ubiquitin ligase gene(Genbank accession no.: MD01 G1010900) was cloned from the Royal Gala apple genome(Malus×domestica Borkh.). Sequence analysis showed that the length of the MdPUB29 gene was 1 275 bp, encoding 424 amino acids. Phylogenetic tree analysis indicated that the apple E3 ubiquitin ligase exhibited the greatest sequence similarity to Pyrus×bretschneideri. The predicted protein structural domain of MdPUB29 showed that it contained a U-box domain. qRT-PCR analysis showed that Md PUB29 was expressed widely in different tissues of the Royal Gala apple species, and was highly expressed in the root, while the expression of MdPUB29 was significantly inhibited by exogenous NaCl. Immunoblotting assays revealed that MdPUB29 protein abundance in tissue cultures of the Royal Gala apple accumulated under NaC l stress conditions. Three-dimensional protein structure prediction indicated that MdPUB29 was highly homologous with AtPUB29. The growing potential of MdPUB29-expressing apple calli and Arabidopsis were much stronger than that of the control under salt stress conditions, suggesting that MdPUB29 may positively regulate salt tolerance.