马氏珠母贝Caspase-3基因克隆和组织表达分析

半胱氨酸蛋白酶3 (Caspase-3)作为细胞凋亡通路中重要的效应蛋白,在细胞凋亡过程中发挥着重要作用.为初步探究马氏珠母贝Caspase-3(PmCaspase-3)的生物学功能,本研究利用cDNA末端快速扩增(RACE)技术克隆获得PmCaspase-3基因cDNA的全长序列并对其序列特征进行分析;同时利用实时荧光定量PCR (RT-qPCR)方法分析了PmCaspase-3基因mRNA在马氏珠母贝不同组织和不同发育时期的表达差异.结果 显示,PmCaspase-3基因cDNA全长为2233 bp,其中5'端非编码区长度为80 bp,3'端非编码长度为31 bp,开放阅读框长度为2088 bp,共编码695个氨基酸;生物信息学分析显示,PmCaspase-3含有Caspase家族特有的CASc结构域和半胱氨酸蛋白酶家族p20、p10活性位点以及多种磷酸化位点,经进化分析以及多序列比对可知与其他物种Caspase-3蛋白同源性较高.RT-qPCR结果表明,PmCaspase-3在肝胰腺中的表达量最高,在闭壳肌中的表达量最低;在发育过程中D型幼虫期和眼点期的表达量较高.     

用中性红标记酵母原生质体初探

用２％蜗牛酶处理酵母细胞６０分钟,啤酒酵母Ｙ２９的原生质体形成率为９０％,再生率为９．５％;糖化酵母ＩＢ的原生质体形成率为８６％,再生率为１２％。用５００ｐｐｍ中性红染液对Ｙ２９菌株的整细胞和原生质体染色１５分钟,细胞的着色率为８４％,存活率为１２％,而原生质体的着色率为７５％,再生率为６．４％,经染色后的原生质体体积缩小,在交变电场中排队所需的场强电降低。     

水稻脆秆突变体bc-s1的表型分析和基因定位

茎秆机械强度影响植株抗倒伏能力, 是备受关注的重要农艺性状之一。与野生型相比, 水稻(Oryza sativa)脆秆隐性突变体bc-s1茎秆抗折力和抗张力分别降低31.1%和67.2%, 茎秆纤维素和木质素含量分别降低24.97%和增高38.82%。细胞学分析显示, bc-s1茎秆厚壁细胞发生不规则变化, 次生壁增厚受阻。通过图位克隆和测序分析, 初步确定bc-s1突变体中纤维素合成酶催化亚基Os09g25490/OsCesA9基因第1外显子的第28个碱基G突变为A。该等位突变体的获得为进一步揭示OsCesA9调控细胞壁建成的生物学功能提供了新的研究材料。     

A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea.     

Disruption of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsulinemia

In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and site-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a beta-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.     

土壤活性有机碳的表征及其生态效应

土壤活性有机碳指在一定的时空条件下,受植物、微生物影响强烈、具有一定溶解性、在土壤中移动比较快、不稳定、易氧化、分解、易矿化,其形态,空间位置对植物、微生物来说活性比较高的那一部分土壤碳素。国外描述这一部分碳素的术语为有效碳、水溶性碳、易氧化碳、可矿...     

Roles of PsbI and PsbM in photosystem II dimer formation and stability studied by deletion mutagenesis and X-ray crystallography

PsbM and PsbI are two low molecular weight subunits of photosystem II (PSII), with PsbM being located in the center, and PsbI in the periphery, of the PSII dimer. In order to study the functions of these two subunits from a structural point of view, we crystallized and analyzed the crystal structure of PSII dimers from two mutants lacking either PsbM or PsbI. Our results confirmed the location of these two subunits in the current crystal structure, as well as their absence in the respective mutants. The relative contents of PSII dimers were found to be decreased in both mutants, with a concomitant increase in the amount of PSII monomers, suggesting a destabilization of PSII dimers in both of the mutants. On the other hand, the accumulation level of the overall PSII complexes in the two mutants was similar to that in the wild-type strain. Treatment of purified PSII dimers with lauryldimethylamine N-oxide at an elevated temperature preferentially disintegrated the dimers from the PsbM deletion mutant into monomers and CP43-less monomers, whereas no significant degradation of the dimers was observed from the PsbI deletion mutant. These results indicate that although both PsbM and PsbI are required for the efficient formation and stability of PSII dimers in vivo, they have different roles, namely, PsbM is required directly for the formation of dimers and its absence led to the instability of the dimers accumulated. On the other hand, PsbI is required in the assembly process of PSII dimers in vivo; once the dimers are formed, PsbI was no longer required for its stability.     

Enantioselective hydrolysis of (R)-2, 2-dimethylcyclopropane carboxamide by immobilized cells of an R-amidase-producing bacterium, Delftia tsuruhatensis CCTCC M 205114, on an alginate capsule carrier

Immobilized cells of Delftia tsuruhatensis CCTCC M 205114 harboring R-amidase were applied in asymmetric hydrolysis of (R)-2, 2-dimethylcyclopropane carboxamide (R − 1) from racemic (R, S)-2, 2-dimethylcyclopropane carboxamide to accumulate (S)-2, 2-dimethylcyclopropane carboxamide (S − 1). Maximum R-amidase activity of 13.1 U/g wet cells (0.982 U/g beads) was obtained under conditions of 3% sodium alginate, 2.5% CaCl₂, 15 h crosslinking and 2 mm bead size, which was 53.9% of that of free cells (24.3 U/g wet cells). In addition, characterization of the immobilized cells was examined. The optimum R − 1 hydrolysis conditions were identified as follows: substrate concentration 10 mM, pH 8.5, temperature 35°C and time course 40 min. Under optimum conditions, the maximum yield and enantiomeric excess of (R)-2, 2-dimethylcyclopropanecarboxylic acid were 49.5% and >99%, respectively. This afforded S − 1 with a yield >49% and an e.e. of 97.7%. With good operational stability and excellent enanotioselectivity, the immobilized cells could be potentially utilized in industrial production of S − 1.     

The Asparaginyl Endopeptidase Legumain Is Essential for Functional Recovery after Spinal Cord Injury in Adult Zebrafish

Unlike mammals, adult zebrafish are capable of regenerating severed axons and regaining locomotor function after spinal cord injury. A key factor for this regenerative capacity is the innate ability of neurons to re-express growth-associated genes and regrow their axons after injury in a permissive environment. By microarray analysis, we have previously shown that the expression of legumain (also known as asparaginyl endopeptidase) is upregulated after complete transection of the spinal cord. In situ hybridization showed upregulation of legumain expression in neurons of regenerative nuclei during the phase of axon regrowth/sprouting after spinal cord injury. Upregulation of Legumain protein expression was confirmed by immunohistochemistry. Interestingly, upregulation of legumain expression was also observed in macrophages/microglia and neurons in the spinal cord caudal to the lesion site after injury. The role of legumain in locomotor function after spinal cord injury was tested by reducing Legumain expression by application of anti-sense morpholino oligonucleotides. Using two independent anti-sense morpholinos, locomotor recovery and axonal regrowth were impaired when compared with a standard control morpholino. We conclude that upregulation of legumain expression after spinal cord injury in the adult zebrafish is an essential component of the capacity of injured neurons to regrow their axons. Another feature contributing to functional recovery implicates upregulation of legumain expression in the spinal cord caudal to the injury site. In conclusion, we established for the first time a function for an unusual protease, the asparaginyl endopeptidase, in the nervous system. This study is also the first to demonstrate the importance of legumain for repair of an injured adult central nervous system of a spontaneously regenerating vertebrate and is expected to yield insights into its potential in nervous system regeneration in mammals.     

基因组改组及其在木质纤维素水解液乙醇发酵菌种选育中的应用展望

能高效代谢木质纤维素水解液中的可发酵糖、同时可耐受/分解发酵抑制剂的菌种, 是利用木质纤维素为原料生产燃料乙醇技术的关键。基因组改组技术是近些年发展起来的一项新型育种技术, 该技术已运用于食品和医药行业菌种的改良。本文综述了基因组改组技术的原理、方法、特点、及其运用, 并对其在木质纤维素水解液乙醇发酵菌种选育方面的应用进行了展望。