玉米遗传转化与商业化转基因玉米开发*

玉米是世界上种植面积最大的粮食作物,为了提高玉米产量和满足人类需求,转基因育种已经成为改良玉米性状的有效手段。自1996年美国种植商业化转基因玉米以来,利用玉米遗传转化技术开发商业化转基因玉米已取得巨大成功。综述了玉米遗传转化体系优化的重要步骤,系统总结了已开发的商业化转基因玉米的种类,并对玉米遗传转化未来发展方向进行了展望。     

抗菌肽Cec4a的重组表达和抗菌活性研究*

目的:构建Cec4a的原核重组表达体系,通过诱导表达、酶切纯化获得重组蛋白,并检测产物的抗菌活性。方法:基于Cec4a的序列设计引物,克隆Cec4a基因的DNA片段。利用原核表达载体(pCold-SUMO)构建重组原核表达质粒,并将其转化到大肠杆菌C41(DE3)等感受态细胞,使用IPTG进行诱导表达。通过Ni-NTA亲和层析柱纯化,获得含有His-SUMO标签的重组Cec4a融合蛋白。在SUMO蛋白酶酶切后,再次使用Ni-NTA亲和层析纯化,得到目的蛋白,最后用鲍曼不动杆菌(ATCC19606)作为指示菌检测表达产物的抗菌活性。结果:成功构建pCold-SUMO-Cec4a原核表达质粒,测序分析其序列与预期结果一致。Cec4a融合蛋白表达量为42.8mg/L,纯化后的Cec4a重组蛋白对鲍曼不动杆菌的MIC为4 μg/mL。结论:通过原核表达,并经Ni-NTA亲和层析纯化,获得了具有抗菌活性的重组蛋白Cec4a,为研究Cec4a的生物活性、抗菌机制及应用奠定了基础。     

日本结缕草耐逆基因ZjLEA2的克隆及功能探究

植物胚胎发育晚期丰富蛋白(Late embryogenesis abundant)在抵御缺水胁迫中起重要作用.本研究从日本结缕草'Meyer'品种中克隆获得了 1个LEA蛋白基因,命名为ZjLEA2.该基因编码区长501个碱基,推测编码的蛋白质含166个氨基酸,预测分子量17kD,等电点9.10,具有LEA蛋白家族第二组的典型结构特征,亲水性强.基因的系统发育树分析结果显示,ZjLEA2蛋白与耐旱的禾本科草种狗牙根和无芒隐子草的同源基因遗传距离最近.ZjLEA2基因的表达受干旱、低温和高盐胁迫诱导,表达量峰值均出现在胁迫72 h后.转化ZjLEA2基因的酵母细胞对冷冻低温和高盐胁迫的耐受能力明显增强.本研究结果说明ZjLEA2基因具有抗逆功能,在结缕草抗非生物胁迫中后期起作用.     

熊蜂主要病虫害的发生和危害现状

熊蜂以其独有的形态特征与生物学特性,在植物授粉中发挥着十分重要作用.然而受寄生虫、真菌等多种病原体影响,近几十年来全球多个地区熊蜂多样性急剧下降.欧洲地熊蜂Bombus terrestris在全球商业化推广应用,加剧了相关熊蜂病虫害的传播和蔓延,已在多地造成生物入侵危害,导致当地传粉蜂多样性下降.本文综述了熊蜂主要病虫害的种类、危害和传播情况,以期有全面深入的认识,并提出对进口商业化熊蜂的管理措施,为我国本土熊蜂资源保护研究奠定基础.     

Transfection of Eimeria mitis with Yellow Fluorescent Protein as Reporter and the Endogenous Development of the Transgenic Parasite

Background

Advancements have been made in the genetic manipulation of apicomplexan parasites. Both the in vitro transient and in vivo stable transfection of Eimeria tenella have been developed successfully. Herein, we report the transient and stable transfection of Eimeria mitis.

Methods and Findings

Sporozoites of E. mitis transfected with enhanced yellow fluorescent protein (EYFP) expression plasmid were inoculated into chickens via the cloacal route. The recovered fluorescent oocysts were sorted by fluorescence activated cell sorting (FACS) and then passaged 6 generations successively in chickens. The resulting population was analyzed by genome walking and Western blot. The endogenous development of the transgenic E. mitis was observed and its reproduction potential was tested. The stable transfection of E. mitis was developed. Genome walking confirmed the random integration of plasmid DNA into the genome; while Western blot analysis demonstrated the expression of foreign proteins. Constitutive expression of EYFP was observed in all stages of merogony, gametogony and sporogony. The peak of the transgenic oocyst output was delayed by 24 h and the total oocyst reproduction was reduced by 7-fold when compared to the parental strain.

Conclusion

Stable transfection of E. mitis was successfully developed. The expression of foreign antigens in the transgenic parasites will facilitate the development of transgenic E. mitis as a vaccine vector.     

LIVIN和HLA-I类分子在肾透明细胞癌中的表达及其临床意义

目的:探讨凋亡抑制蛋白Livin与人类白细胞抗原(HLA)I类分子在人类肾脏透明细胞癌(clear cell renal cell carcinoma,ccRCC)中的表达及其临床意义。方法:选择哈尔滨医科大学附属第一医院2007年12月至2011年12月收治的拟行肾癌根治术的患者80例,应用免疫组织化学方法检测80例ccRCC组织与10例癌旁正常肾组织中Livin及HLA-I类分子的表达,并分析其与患者临床病理特征之间的关系。结果:10例癌旁正常肾组织中均未见Livin的阳性表达,而HLA-I类分子的阳性表达率为100%。ccRCC组织中,Livin的阳性表达率及HLA I类分子表达下调百分率分别为41(51.25%)例和48(60.00%)例,Livin的阳性表达率与正常组相比差异有统计学差异(P0.05)。ccRCC组织中Livin的阳性表达率与淋巴结转移、组织分化程度显著相关(P0.05),而HLA I类分子的表达下调百分率与患者的性别、年龄、肿瘤分级、淋巴结转移及分期均无相关性(P0.05)。ccRCC组织中Livin的表达与HLA I类分子的表达亦无显著相关性(P0.05)。结论:Livin、HLA I类分子表达异常参与了ccRCC的发生、发展过程,Livin可能成为ccRCC的一个重要预后指标和治疗靶点。     

CD117 Expression in Fibroblasts-Like Stromal Cells Indicates Unfavorable Clinical Outcomes in Ovarian Carcinoma Patients

The stem cell factor (SCF) receptor CD117 (c-kit), is widely used for identification of hematopoietic stem cells and cancer stem cells. Moreover, CD117 expression in carcinoma cells indicates a poor prognosis in a variety of cancers. However the potential expression in tumor microenvironment and the biological and clinical impact are currently not reported. The expression of CD117 was immunohistochemically evaluated in a serial of 242 epithelial ovarian cancer (EOC) cases. Thirty-eight out of 242 cases were CD117 positive in fibroblast-like stromal cells and 22 cases were positive in EOC cells. Four cases were both positive in fibroblast-like stromal cells and EOC cells for CD117. CD117 expression in fibroblast-like stromal cells in ovarian carcinoma was closely linked to advanced FIGO stage, poor differentiation grade and histological subtype (p<0.05), and it was significantly associated with poor overall survival (OS) and progression free survival (PFS) (Kaplan-Meier analysis; p<0.05, log-rank test). CD117 expression in ovarian carcinoma cells was not associated with these clinicopathological variables. The CD117 positive fibroblast-like stromal cells were all positive for mesenchymal stem/stromal cell (MSC) marker CD73 but negative for fibroblast markers fibroblast activation protein (FAP) and α smooth muscle actin (α-SMA), indicating that the CD117+/CD73+ fibroblast-like stromal cells are a subtype of mesenchymal stem cells in tumor stroma, although further characterization of these cells are needed. It is concluded herewith that the presence of CD117+/CD73+ fibroblast-like stromal cells in ovarian carcinoma is an unfavorable clinical outcome indication.     

广西大石围天坑群天坑森林主要木本植物种间关联性研究

为了解大石围天坑群天坑森林植物群落的种间联结特征,该文以大石围天坑群中7个典型天坑森林为研究区建立18个样地,取其中重要值大于1的木本植物为研究对象,采用方差比例、χ2检验、Pearson相关系数检验、Spearman秩相关系数检验及PCA排序方法,对天坑森林内的主要木本植物种间关联性和生态种组进行定量分析。结果表明:(1)乔木层总体联结性呈现显著正联结,灌木层总体联结性呈现不显著正联结,群落总体关联性呈现显著正联结,表明天坑森林植物群落处于稳定的阶段。(2)χ2检验、Pearson相关系数检验和Spearman秩相关系数检验发现,乔木层正联结种对多于负联结种对且显著联结种对占多数,灌木层负联结种对多于正联结种对,且显著联结种对占多数,表明乔木层群落稳定性高于灌木层,乔木层种间竞争合作强,灌木层种间竞争排斥性强,群落主要木本植物之间联结性强,种间保持紧密联结的联结特征。(3) PCA排序将天坑森林植物分为四大生态种组,物种分布由天坑底部至顶部呈现圈层分布方式,且物种分布方式总体受生境差异和物种自身特性影响。可见,植物紧密的联结关系是天坑森林多样性维持机制之一,而群落内部种间关联性的自我调控及物种对生境异质性的趋同适应是群落维持稳定的基础。     

Mutation analysis of the WNT4 gene in Han Chinese women with premature ovarian failure

Background

The WNT4 gene plays an important role in female sex determination and differentiation. It also contributes to maintaining of the ovaries and the survival of follicles.

Methods

We sequenced the coding region and splice sites of WNT4 in 145 Han Chinese women with premature ovarian failure (POF) and 200 healthy controls.

Results

Only one novel variation, in Exon 2 (195C > T), was detected among the women with POF. However, this synonymous variation did not result in a change in amino acid sequence (65 Asp > Asp). No further variants were found in any of the samples.

Conclusion

Although we cannot provide any evidence that it is a possible disease-causing gene, this study is the first attempt to investigate the possible role of WNT4 in Han Chinese women with POF.     

Efficiency and fidelity of human DNA polymerases λ and β during gap-filling DNA synthesis

The base excision repair (BER) pathway coordinates the replacement of 1-10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1-10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5'-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER.