中华按蚊CYP6Y亚家族基因的鉴定和生物信息学分析

【目的】鉴定中华按蚊Anopheles sinensis CYP6Y亚家族基因,分析它们的结构和特征,推测其可能的功能。【方法】以冈比亚按蚊An. gambiae CYP6Y1作为询问序列,通过双向Blast方法检索中华按蚊转录组中CYP6Y亚家族基因,并通过生物信息学方法分析基因结构、特征及可能的功能。【结果】从中华按蚊转录组测序数据中鉴定出2条CYP6Y亚家族基因,分别命名为AsCYP6Y1（GenBank登录号：KF709397）和AsCYP6Y2（GenBank登录号：KF709398）。序列分析显示,AsCYP6Y1和AsCYP6Y2全长分别为1 713 bp和1 815 bp,分别编码502和526个氨基酸。基因结构分析显示,该亚家族基因仅含有1个相位1型内含子并与其他P450基因形成保守的共线性分布。蛋白结构分析显示,这2个基因编码的蛋白含P450特有的5个特征序列和6个底物结合位点,且均不存在信号肽,其亚细胞定位为细胞质。3D结构分析显示,AsCYP6Y1有18条α螺旋和13股反向平行的β折叠,AsCYP6Y2有19条α螺旋和11股反向平行的β折叠。通过同样的方法,在达林按蚊An. darlingi中也鉴定出2个CYP6Y亚家族基因。系统进化分析显示,AsCYP6Y1和AsCYP6Y2分别与其他3种按蚊的CYP6Y1和CYP6Y2聚成一支,Bootstrap值均大于90%。替换率分析显示,中华按蚊AsCYP6Y1和AsCYP6Y2与其他3种按蚊同源基因的Ka/Ks均小于1。相对进化速率分析显示,中华按蚊CYP6Y和CYP6M亚家族的相对进化速率均显著快于CYP6P亚家族,而CYP6Y和CYP6M亚家族之间没有显著差异。【结论】在中华按蚊和达林按蚊中存在2个CYP6Y亚家族基因,之前在冈比亚按蚊和不吉按蚊An. funestus中也发现2个CYP6Y亚家族基因,表明CYP6Y亚家族基因可能在按蚊属广泛存在,且可能为按蚊属昆虫所特有。     

葱蝇海藻糖-6-磷酸合成酶基因的克隆、序列分析及滞育相关表达

海藻糖-6-磷酸合成酶（trehalose-6-phosphate synthase, TPS）是昆虫海藻糖合成途径中的关键酶之一。本研究通过对葱蝇Delia antiqua海藻糖-6-磷酸合成酶基因的克隆、 序列分析及滞育相关表达的分析, 旨在证明该基因在能源合成以及抵御高温和低温环境方面发挥重要作用, 为进一步弄清葱蝇滞育分子机制提供理论依据。根据葱蝇抑制消减杂交文库中的EST序列信息, 设计特异性引物, 并通过RACE技术克隆了葱蝇海藻糖-6-磷酸合成酶基因全长cDNA, 命名为DaTPS1 （GenBank登录号： JX681124）, 其全长为2 904 bp, 开放阅读框2 448 bp, 编码815个氨基酸, 推测其相对分子质量为91.2 kD, 等电点为5.96。生物信息学分析表明, 该基因编码的氨基酸序列具有两个保守结构域, 与其他物种TPS具有较高的同源性, 其中和黑腹果蝇Drosophila melanogaster亲缘关系最近, 氨基酸序列一致性为92.1%; 其蛋白质三维结构有15条大的α螺旋和11股反向平行的β链折叠。RT-PCR分析表明, DaTPS1在葱蝇非滞育、 夏滞育和冬滞育期蛹中都有表达, 但是非滞育期各时期表达量基本没有变化, 而在夏滞育和冬滞育蛹的滞育前期表达量较高, 滞育保持期表达量较低, 滞育期后期表达量又有所升高。推断在葱蝇蛹夏滞育和冬滞育期前期, TPS1开始催化合成较多的海藻糖以提高滞育期抵御不良环境的能力, 滞育保持期蛹的新陈代谢降低, 所需能量较少, 所以TPS1处于低水平表达状态, 而滞育期结束后, 蛹生长发育逐渐恢复, 所需能量有所增加, TPS1的表达量再次升高。本研究对揭示昆虫TPS在能量代谢通路中的作用及昆虫滞育的分子机理具有一定的科学意义。     

稻瘿蚊种群DNA指纹及水稻抗稻瘿蚊分子标记育种技术研究

文章综述经10年(1996~2005)研究建立的亚洲稻瘿蚊Orseolia oryzae Wood-Mason种群DNA指纹检测技术和水稻抗稻瘿蚊分子育种技术的研究成果。其核心技术包括5点:(1)在国际和国内首先用AFLP方法研究亚洲5国(中国,印度,斯里兰卡,尼泊尔和老挝)及广东省7个地点亚洲稻瘿蚊种群DNA指纹及其生物多样性;(2)分别用RAPD和微卫星SSR技术对来源于我国的水稻资源大秋其的抗亚洲稻瘿蚊基因Gm6进行精细定位;(3)分别用与Gm6基因紧密连锁的STS分子标记和SSR标记,建立了分子标记辅助选择(MAS)方法选育抗稻瘿蚊新品种的技术体系;(4)应用建立的分子标记辅助选育新技术选育出一批抗亚洲稻瘿蚊中国种群的新品系,创造出一批新种质,包括抗蚊18号、抗蚊软占等达国优3级的抗稻瘿蚊新品系6个;(5)将Gm6基因导入杂交稻恢复系,选育出4个抗稻瘿蚊两系杂交稻新组合培矮64S/KG18,培矮64S/KI41,培矮64S/AK7,培矮64S/03W16(国优2级)和1个三系杂交稻新组合抗蚊博优。其中江西省宁都市名林水稻研究所应用本研究鉴定出的带有Gm6基因的品系抗蚊青占作为抗性亲本,培育出的2个抗稻瘿蚊的两系杂交稻组合安两优青占和培两优抗占通过省级品种审定,率先选育出抗稻瘿蚊种群的杂交稻。该项研究成果为控制华南稻瘿蚊的灾害发生提供新的技术措施,选育的抗稻瘿蚊品种和新种质,在华南广为应用,取得显著的经济效益。     

棉铃虫Hsp70的多克隆抗体制备及鉴定

HSPs(热休克蛋白)是机体在不利环境条件刺激下合成的一类蛋白质,在进化上高度保守,普遍存在于生物体,其中Hsp70是最为保守的成员。研究发现,昆虫滞育过程中普遍存在Hsp70表达上调的现象。然而,现有的研究均是在基因水平的检测结果。为了能从蛋白水平检测棉铃虫中Hsp70的表达,本研究制备了棉铃虫Hsp70的多克隆抗体。构建了hsp70的原核表达载体并在大肠杆菌BL21(DE3)中成功表达,重组蛋白经镍柱纯化后免疫兔子,制备了棉铃虫Hsp70的多克隆抗体。抗体的效价较高,达到了1∶256 000,此外Western结果表明,制备的抗体能检测出热诱导的Hsp70蛋白。本研究制备了高效价且较为特异的Hsp70对克隆抗体,该抗体为后续从蛋白水平研究棉铃虫滞育过程中Hsp70的表达及其分子机制奠定了基础。     

外泌体环状RNA在肿瘤微环境中的研究进展

外泌体是细胞分泌的30～150 nm的细胞外囊泡,在肿瘤微环境(tumor microenvironment,TME)中介导细胞间通讯.环状RNA (circular RNA,circRNAs)是一类由前体mRNA (precursor mRNA,pre-mRNA)反向剪接生成的非编码RNA(non-coding RNA,ncRNA),在外泌体中富集且表达稳定.本文主要讨论外泌体起源和circRNAs在外泌体中的分选调控机制,阐述外泌体circRNAs在肿瘤微环境各个阶段中的作用与机制,包括血管生成、EMT、耐药等.最后,本文探讨外泌体circRNAs作为肿瘤标志物和治疗靶点的临床应用前景与价值.     

短发夹 RNA 介导 RNA 干扰的时间 和剂量效应研究

用 RNA 干扰 (RNA interference , RNAi) 技术抑制哺乳动物细胞中外源报告基因的表达,以探讨该过程中 RNAi 作用的剂量和时间效应 . 应用 Lipofectamine 2000 将外源报告基因的表达载体与编码短发夹 RNA (short hairpin RNA , shRNA) 的质粒共转染 HEK293H 细胞,观察 shRNA 载体对报告基因的抑制效应 . 转染后, shRNAs 的瞬时表达可特异地抑制细胞内报告基因的表达 . 在共转染后 12 , 24 , 48 , 60 , 72 , 96 h 时检测 EGFP (enhanced green fluorescent protein , EGFP) 基因 mRNA 及蛋白质表达水平,结果显示, EGFP mRNA 及蛋白质表达在 12 h 时略有降低, 24~48 h 时表达逐渐降低, 48~72 h 时降低最明显,其后 EGFP 表达水平逐渐恢复 . 提示该过程中 RNAi 效应呈现由弱到强、又由强到弱的逐渐消逝趋势 . 共转染一系列剂量比例的 EGFP 干扰载体与靶载体的结果表明,在一定剂量范围内, RNA 干扰载体所介导的抑制效应与干扰载体剂量大小有关,当其剂量进一步加大足以抑制外源基因表达时,抑制效应则维持在一“平台期” . 此外,通过 RNAi 抑制 HeLa 细胞、 HEK293 细胞中荧光素酶基因的表达, 荧光素酶活性变化也表现出上述类似的效应 . 这些结果表明,在体外哺乳动物细胞中,基于表达载体的 RNAi 作用呈现剂量和时间依赖性效应 . 这为基于载体表达的 RNAi 技术应用研究提供了一定的理论参考及依据 .     

Association between low density lipoprotein receptor-related protein 2 gene polymorphisms and bone mineral density variation in Chinese population

Low density lipoprotein receptor-related protein 2 gene (LRP2) is located next to the genomic region showing suggestive linkage with both hip and wrist bone mineral density (BMD) phenotypes. LRP2 knockout mice showed severe vitamin D deficiency and bone disease, indicating the involvement of LRP2 in the preservation of vitamin D metabolites and delivery of the precursor to the kidney for the generation of 1α,25(OH)(2)D(3). In order to investigate the contribution of LRP2 gene polymorphisms to the variation of BMD in Chinese population, a total of 330 Chinese female-offspring nuclear families with 1088 individuals and 400 Chinese male-offspring nuclear families with 1215 individuals were genotyped at six tagSNPs of the LRP2 gene (rs2389557, rs2544381, rs7600336, rs10210408, rs2075252 and rs4667591). BMD values at the lumbar spine 1-4 (L1-4) and hip sites were measured by DXA. The association between LRP2 polymorphisms and BMD phenotypes was assessed by quantitative transmission disequilibrium tests (QTDTs) in female- and male-offspring nuclear families separately. In the female-offspring nuclear families, rs2075252 and haplotype GA of rs4667591 and rs2075252 were identified in the nominally significant total association with peak BMD at L1-4; however, no significant within-family association was found between peak BMD at the L1-4 and hip sites and six tagSNPs or haplotypes. In male-offspring nuclear families, neither the six tagSNPs nor the haplotypes was in total association or within-family association with the peak BMD variation at the L1-4 and hip sites by QTDT analysis. Our findings suggested that the polymorphisms of LRP2 gene is not a major factor that contributes to the peak BMD variation in Chinese population.     

Surveillance on the Status of Immune Cells after Echinnococcus granulosus Protoscoleces Infection in Balb/c Mice

Background

Cystic echinococcosis is a global parasitic disease caused by infection with Echinococcus granulosus larvae with potentially life-threatening complications in humans. To date, the status of the immune cells believed to be associated with the pathogenicity of E. granulosus infection has not been demonstrated clearly.

Methodology/Principal Findings

In this study, we developed a multiplex flow cytometry assay to investigate the systemic immune status of innate and adaptive immunity at 30, 180, 360 days post-infection (dpi) in mice infected with E. granulousus. At 30 dpi, an increase in the number of CD11b⁺ and CD11c⁺ antigen-presenting cells (APCs) was observed. This was accompanied by the slight down-regulated expression of the co-stimulatory molecule MHC-II, indicating the impairment of APCs in early infection through the release of secretory-excretory products. In all infected groups, we observed a significant increase in innate immune cells, including APCs and GR-1⁺ cells, and a dramatic increase in the myeloid-derived suppressor cells (MDSC) expressing CD11b⁺/GR-1⁺. Moreover, the upregulation of the activated markers CD69, CD44, CD40L, and the downregulation of CD62L were observed in the CD4⁺ and CD8⁺ T cells following infection. Regulatory T cells expressing CD4⁺/CD25⁺/FoxP₃ ⁺ increased significantly over the course of infection.

Conclusions

Our findings demonstrate that the microenvironment in the peripheral immune system after E. granulosus infection changes in subtle but detectably ways, especially during the persistent period of infection. We found that T cells were activated following infection, but observed that the significant increase of immunosuppressive cells such as MDSC and Treg cells could inhibit T cell response to E. granulosus antigens. We suggest these cells may play a neglected but key role in the downregulation of the immune response in long-term parasitic infection. Understanding the basic functions and temporal interactions of these immunosuppressive cells will pave the way for new strategies of parasite vaccine design.     

Contribution of common deletion to total deletion burden in mitochondrial DNA from inner ear of d-galactose-induced aging rats

Mitochondrial DNA (mtDNA) mutations, especially deletions, have been suggested to play an important role in aging and degenerative diseases. In particular, the common deletion in humans and rats (4977bp and 4834bp deletion, respectively) has been shown to accumulate with age in post-mitotic tissues with high energetic demands. Among numerous deletions, the common deletion has been proposed to serve as a molecular marker for aging and play a critical role in presbyacusis. However, so far no previous publication has quantified the contribution of common deletion to the total burden of mtDNA deletions in tissues during aging process. In the present study, we established a rat model with various degrees of aging in inner ear induced by three different doses of d-galactose (d-gal) administration. Firstly, multiple mtDNA deletions in inner ear were detected by nested PCR and long range PCR. In addition to the common deletion, three novel mtDNA deletions were identified. All four deletions, located in the major arc of mtDNA, are flanked by direct repeats and involve the cytochrome c oxidase (COX) subunit III gene, encoded by mtDNA. Additionally, absolute quantitative real-time PCR assay was used to detect the level of common deletion and total deletion burden of mtDNA. The quantitative data show that the common deletion is the most frequent type of mtDNA deletions, exceeding 67.86% of the total deletion burden. Finally, increased mtDNA copy number, reduced COX activity and mosaic ultrastructural impairments in inner ear were identified in d-gal-induced aging rats. The increase of mtDNA replication may contribute to the accelerated accumulation of mtDNA deletions, which may result in impairment of mitochondrial function in inner ear. Taken together, these findings suggest that the common deletion may serve as an ideal molecular marker to assess the mtDNA damage in inner ear during aging.