费氏丙酸杆菌谢氏亚种和植物乳杆菌中亚油酸异构酶的分离纯化

采用盐析、Sephacryl S-200HR凝胶过滤层析、DEAE Sepharose F.F.离子交换层析相结合的方法,分别建立了来源于费氏丙酸杆菌谢氏亚种（P. feudenreichii ssp. shermanii）和植物乳杆菌（L. plantarum）的亚油酸异构酶的分离提取步骤。结果表明不同来源的亚油酸异构酶在纯化的过程中表现出不同的洗脱特性,P. freudenreichii ssp. shermanii亚油酸异构酶的分子质量约为56 kDa, L. plantarum亚油酸异构酶的分子质量为50.65 kDa。不同来源亚油酸异构酶的分离纯化方法存在差异。     

A transgenic durum wheat line that is free of marker genes and expresses 1Dy10

As currently practiced, genetic engineering of monocots requires the use of selective agents, such as herbicides and antibiotics, and marker genes for resistance to favor the multiplication of the initially transformed cells. In the present paper we have used “minimal gene cassettes” and positive selection to generate transgenic durum wheat lines free of herbicide and antibiotic resistance marker genes. Two biolistic transformation experiments were carried out using three “minimal gene cassettes” consisting of linear DNA fragments each excised from the source plasmids. The targeted trait genes were two bread wheat sequences encoding the Dx5 and Dy10 high-molecular-weight (HMW) glutenin subunits, which have been associated with superior bread-making quality and which are absent from durum wheats. The positive selectable marker was the Escherichia coli phosphomannose isomerase (pmi) gene, whose product catalyzes the reversible interconversion of mannose-6-phosphate and fructose-6-phosphate, allowing plant cells to utilize mannose as a carbon source. PCR assays of genomic DNA from regenerated plants identified 15 T₀ plants that contained the pmi marker gene for an overall transformation efficiency of 1.5%, which is similar to biolistic transformation efficiencies of durum wheat with intact circular plasmids. Line TC-52, which initially contained pmi, non-expressed 1Dx5, and expressed 1Dy10 HMW glutenin subunit transgenes, was further investigated. PCR was used to follow inheritance of the pmi marker gene and 1Dx5 from the T₁ to T₃ generations. Transgene expression was monitored by the chlorophenol-red assay for pmi and SDS-PAGE of seed proteins for 1Dy10. From these analyses, we observed that the 1Dy10, 1Dx5 and pmi transgenes were not linked, allowing us in the T₃ generation to identify 1Dy10 transgenic segregants that contained no marker or silent 1Dx5 transgenes. Homozygotes containing and expressing only the 1Dy10 transgene were identified in the T₄ generation. These experiments show that it is possible to combine biolistic transformation by minimal gene cassettes with genetic segregation to make marker-free transgenic wheat plants with new traits.     

拟南芥木糖异构酶在酿酒酵母的成功表达

酿酒酵母是工业上乙醇发酵的首选目标微生物,但是不能代谢木糖等五碳糖.通过导入外源木糖异构酶基因,可以赋予酵母利用木糖的能力.但是长期的研究发现很难将细菌木糖异构酶转入酿酒酵母得到活性表达.研究成功的在酿酒酵母里表达了拟南芥木糖异构酶,其活性可达到0.51 U/(mg·protein).拟南芥木糖异构酶与成功在酵母里表达的真菌木糖异构酶相比更加耐受木糖醇抑制(Ki=13.21 mM).本研究为改善酵母利用木糖提供了新的可用的木糖异构酶.     

紫花苜蓿蛋白质二硫键异构酶mPDI的生物信息学分析与同源建模

紫花苜蓿蛋白质二硫键异构酶的基因已经被克隆测序。利用其mRNA及氨基酸序列,应用生物信息学软件预测了该蛋白质的理化性质、亲/疏水性、信号肽、二级结构、卷曲螺旋结构、跨膜区域、糖基化位点、活性位点、亚细胞定位、功能结构域及高级结构。结果表明,该蛋白质是一个整体疏水性蛋白,细胞定位为粗面内质网,含有512个氨基酸,理论等电点为4.98,信号肽位于1～24号氨基酸;二级结构中α-螺旋占26.37％（135AA）,无规则卷曲占53.32％（273AA）,延伸链占20.31％（104AA）;包含3个卷曲螺旋结构,3个糖基化位点,2个硫氧还蛋白结构域,2个硫氧还蛋白活性位点。Ramachandram结构检测表明此模型的三维结构符合立体化学能量规则。     

紫参薯查尔酮合成酶及异构酶基因的克隆与表达分析

查尔酮合成酶及其异构酶是黄酮类化合物合成途径中的2个关键酶,为研究其在紫参薯花青素合成途径中的功能,利用RT-PCR技术从紫参薯Da56块茎中克隆到查尔酮合成酶(chalcone synthase,CHS)和查尔酮异构酶(chalcone isomerase,CHI)基因,分别命名为DaCHS和DaCHI。结果表明:DaCHS基因包含825 bp的完整开放阅读框(ORF),预测编码274个氨基酸;DaCHI基因包含663 bp的完整开放阅读框,预测编码221个氨基酸。生物信息学分析结果表明,紫参薯DaCHS与油棕Eg CHS亲缘关系最近,氨基酸相似性达到90%;紫参薯DaCHI与野茶树CaCHI亲缘关系最近,氨基酸相似性达到67%。实时荧光定量PCR检测结果表明,DaCHS和DaCHI基因均具有组织表达特异性,其中DaCHS在花中相对表达量最高,而DaCHI在块茎中相对表达量最高;在紫参薯块茎发育不同时期的表达分析表明,DaCHS、DaCHI均在薯块膨大期相对表达量最高。本研究通过对DaCHS和DaCHI基因全长cDNA序列的克隆与分析,为紫参薯花青素合成途径的遗传调控及花青素形成机理的研究奠定基础。     

Immunization with mixed peptides derived from glucose-6-phosphate isomerase induces rheumatoid arthritis in DBA/1 mice

AIM: To establish an animal model of rheumatoid arthritis(RA) in DBA/1 mice induced by immunodominant mixed peptides derived from glucose-6-phosphate isomerase(GPI). METHODS: The DBA/1 mice were immunized with emulsified mixed peptide fragments of hGPI325-339+hGPI469-483 or single peptide hGPI325-339 in complete Freund's adjuvant by subcutaneous injection to induce the model of RA. Body weight, ankle joint symptom scores, the pathological change of the ankle joint, the levels of CD4⁺ T cells in the spleen and peripheral blood, the proportion of iNKT cells in the peripheral blood, and the levels of TNF-α and IL-6 in serum were detected to evaluate and analyze the model. RESULTS: The hind paw of the model mice appeared red swelling on the 8th day, and then aggravated gradually to the limbs. The red swelling reached peak on the 14th day, and then relieved gradually. Inflammation response dominated by lymphocytes and monocytes was observed in the ankle joint. The inflammatory effect of mixed peptides was more obvious than that of the single one(P<0.05). Compared with control group and the mice treated with single peptide, the weight gain was slow, the amount of CD4⁺ T cells in the peripheral blood and spleen were increased, the proportion of peripheral iNKT cells in the inflammatory peak was decreased(P<0.05), and the serum level of TNF-α was increased significantly(P<0.05) in the mice treated with mixed peptide fragments. CONCLUSION: The immunological characteristics of RA model induced by mixed GPI peptides in DBA/1 mice is closer to that in RA patients, especially in the immunopathology of iNKT cells. Therefore, this model can be used as a new tool for studying the mechanism and immunological intervention of RA.     

mgr-mir-9 implicates Meloidogyne graminicola infection in rice by targeting the effector MgPDI

MicroRNAs (miRNAs), a class of small non-coding RNAs, are crucial endogenous gene regulators in a range of animals, including plant-parasitic nematodes. Meloidogyne graminicola is an obligate sedentary endoparasite of rice and causes significant yield losses. A number of studies focused on the roles of M. graminicola effectors during the parasitic process; however, how nematode miRNAs regulate its effectors needs elucidating. In this research, we analyzed a cluster of M. graminicola miRNAs obtained at the second-stage juveniles (J2s) stage that are closely linked to the regulation of M. graminicola effectors. There are 49 767 105 total clean reads obtained from three libraries. A total of 233 known miRNAs and 21 novel miRNAs were identified. Among the known miRNAs, mgr-lin-4, mgr-mir-1, mgr-mir-100, mgr-mir-86, mgr-mir-279, mgr-mir-87, mgr-mir-71, mgr-mir-9, mgr-mir-50, mgr-mir-72, and mgr-mir-34 are the most abundant 11 miRNAs families. Moreover, the expression levels of selected miRNAs were validated by real-time quantitative PCR. We hypothesized that these miRNAs might regulate the expression of secreted effectors during the J2s stage to facilitate its infection. Consistent with this, we found that mgr-mir-9 targets MgPDI, an important M. graminicola effector mRNA. In addition to that, J2s treated with mgr-mir-9 mimics showed down-regulation of MgPDI expression and reduced reproductive ability, alluding mgr-mir-9 is involved in nematode infection. These results provide novel insight into the regulatory functions of M. graminicola miRNAs during the infection and identify miRNAs and their effector targets as potential key management targets to limit parasite survival during the early stages of infection.     

棉花查尔酮异构酶基因家族的鉴定及表达分析

为研究陆地棉查尔酮异构酶CHI（chalcone isomerase）在彩色棉纤维发育及响应胁迫中的作用，对陆地棉基因组中GhCHI基因家族进行鉴定，开展蛋白理化性质、结构域、启动子顺式作用元件、蛋白质高级结构、系统进化分析，利用转录组数据和实时荧光定量PCR分析其表达特征。结果显示，从陆地棉基因组中鉴定获得12个GhCHI基因家族成员，可分为2个亚家族，具有典型的查尔酮超家族结构域，编码201~452个氨基酸，主要为亲水性蛋白，二级结构主要以α-螺旋和无规则卷曲为主。GhCHI基因启动子顺式作用元件类型主要包含光反应元件、激素响应元件及参与类黄酮生物合成调控的元件等。表达分析结果显示GhCHI1、GhCHI2、GhCHI3、GhCHI4与纤维发育密切相关，尤其在彩色棉纤维发育后期的色素沉积着色时期表达水平较高；这4个基因在叶、花托、雌蕊中也具有较高的表达水平；GhCHI1、GhCHI2、GhCHI3在热胁迫、盐胁迫和干旱胁迫下均受到显著的诱导表达。GhCHI1―GhCHI4蛋白互作分析结果显示，它们可能和参与类黄酮合成的2-氧代戊二酸3-双加氧酶和类黄酮3''-单加氧酶等蛋白质存在相互作用。结果表明，GhCHI1、GhCHI2、GhCHI3和GhCHI4基因在彩色棉的纤维发育和胁迫应答中发挥重要作用。