锌指核酸酶技术在植物基因工程中的应用

锌指核酸酶(zinc finger nucleases,ZFNs)由3到4个锌指结构(zinc fingers,ZFs)和FokⅠ核酸内切酶的剪切结构域组成。锌指核酸酶(ZFNs)通过锌指结构(ZFs)与特异核酸位点结合,再利用FokⅠ的酶切作用切割DNA,引起特异位点DNA双链断裂(double strand break,DSB)。DNA双链断裂可以通过非同源末端连接(non-homologous end joining,NHEJ) 或同源重组(homologous recombination,HR)来修复。在修复过程中实现对基因组DNA的靶向修饰。介绍了锌指核酸酶结构、人工构建途径,作用机理和试验步骤,重点综述了锌指核酸酶技术在植物基因工程的应用。     

肾移植术后HCMV感染与淋巴细胞亚群及肾功能的相关性

目的：肾移植患者由于术前透析及术后服用免疫抑制剂,显著增加了人巨细胞病毒（Humancytomegalovirus,HCMV）原发感染和潜伏感染被激活的机会。观察HCMV感染情况与血T淋巴细胞亚群及肾功能的变化,以探讨其相关性及临床意义。方法：跟踪收集40例肾移植患者术前、术后血标本,应用RT-PCR技术检测HCMV,流式细胞术检测淋巴细胞亚群,结合肾功能判断是否发生急性排斥或移植肾功能恢复延迟。结果：肾移植术后HCMV感染率为52．5％,10例出现症状性感染（25％）,出现阳性时间35．7±15．3天。症状性感染组CD3-bCD4＋的水平和CD4＋／CD8＋的比值较无活动性感染组和正常对照组均降低,CD8＋水平较其他组升高,差异有显著性意义（P〈0．05）。无症状性活动感染者与无活动性感染者各指标比较差异无显著性。活动性感染组急性排斥或移植肾功能恢复延迟发生8例（38．1％）,无活动性感染组发生1例（5．2％）,差异具有统计学意义（x^2=6．15,P〈0．05）。结论：肾移植术后HCMV感染能显著引起T淋巴细胞亚群变化,尤其是CD4＋／CD8＋。并与急性排斥或移植肾功能恢复延迟密切相关联,在其发生发展中可能起着重要的作用。三者相互联系、相互作用、互为因果,对进一步机制的研究及临床诊断治疗、预后方面有一定意义。     

药用真菌猪苓溶血素基因克隆和序列分析

利用3'-RACE-PCR方法首次从药用真菌猪苓中克隆得到与真菌形态发育相关的溶血素基因。结果表明,猪苓溶血素基因的全长cDNA为744bp,其中编码区占447bp,共编码148个氨基酸,推测其分子量约为15.79kDa,理论等电点为4.89。推定的猪苓溶血素蛋白具有与杨树菇溶血素类蛋白家族相同的结构域和功能位点,两者同源性为60%。系统进化树结果显示猪苓溶血素隶属于担子菌类群。实时荧光定量PCR分析结果表明在菌核形成初期猪苓溶血素基因表达量较高,且显著高于菌丝体中猪苓溶血素基因的转录水平,说明溶血素基因参与了猪苓菌核的形态发育。     

川明参果实发育过程中解剖结构的变化

对伞形科(Apiaceae)中国特有单型属种类川明参（Chuanminshen violaceum Sheh et Shan）幼果发育阶段（包含幼果期Ⅰ、幼果期Ⅱ和幼果期Ⅲ）、中果发育阶段（包含中果期Ⅰ、中果期Ⅱ和中果期Ⅲ）和熟果发育阶段（包含熟果期Ⅰ、熟果期Ⅱ和熟果期Ⅲ）果实横切面的解剖结构进行了比较观察。观察结果表明,川明参果实在果壁细胞层数和形态、果棱和维管束、油管、胚乳以及分生果横切面大小和形状等特征均有明显的变化。川明参分生果果壁由厚变薄且外果壁、中果壁细胞形态发生变化,内果壁细胞消失;果实中油管分布在果实的背面、侧面和合生面,油管不断向外扩大,在成熟果实中油管往往皱缩。川明参果实中胚乳逐渐增大,从近球形变化为桃形、肾形直至月牙形,背部呈山丘状隆起;分生果横切面的形状由近五边状圆形变化至梭形,背腹极度压扁。通过对果实发育形态解剖观察发现,果实发育前期,果形的变化主要依靠维管束的发育,从而造成果棱的改变,来改变果实外部结构。到了熟果期,胚乳显现,形体变大,它的形状变化开始改变分生果的外部形态,并且影响较为显著。     

The effects of different vegetation restoration patterns on soil bacterial diversity for sandy land in Hulunbeier

Grassland desertification seriously threatens sustainable economic and social development. Much attention has been paid to the control of grassland desertification, and even to the restoration and reconstruction of the grassland. Vegetation restoration is considered to be a very effective solution. Soil sustains an immense diversity of microbes, and the characteristics of soil microbial communities are sensitive indicators of soil. It is important to understand the relationship between vegetation and soil microbial diversity during the restoration process. Soil microbial, which is the main index to evaluate soil quality, plays a significant role in ecosystem and soil microbial diversity is the important one of global diversity. Exploring the effects of different vegetation patterns on soil microbial diversity can provide scientific bases and technical support for systemic and impersonal assessment of the best vegetation restoration patterns, as well as the vegetation restoration and reconstruction of Hulunbeier sandy land. Based on PCR–DGGE technology, a case study was carried out to investigate the effects of five different vegetation restoration patterns on soil microbial functional diversity after 4 years in sandy land in Hulunbeier, China. The five vegetation restoration patterns included mono-cultivar planting of Agropyron cristatum (UA), mono-cultivar planting of Hedysarum fruticosum (UH), mono-cultivar planting of Caragana korshinskii (UC), mixed-cultivar planting of Agropyron cristatum and Hedysarum fruticosum (AC) and mixed-cultivar planting of Agropyron cristatum, Hedysarum fruticosum, Caragana korshinskii and Elymus nutans (ACHE). Completely degraded sandy land was used as control.The results indicated that the vegetation restoration increased the genetic diversity of soil bacterial community obviously, and the structure of soil bacterial community was changed. The results of phylogenetic analysis suggested that the bacterial community in Hulunbeier sandy land mainly attributed to Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, and Acidobacteria. The dominant groups were Proteobacteria and Bacteroidetes. The effects of different vegetation type on soil bacterial community structures were different.     

Association between Vitamin D Insufficiency and Elevated Serum Uric Acid among Middle-Aged and Elderly Chinese Han Women

Background

Association between vitamin D insufficiency and hyperuricemia has not been reported so far. We aimed to study the association of vitamin D insufficiency with elevated serum uric acid among middle-aged and elderly Chinese Han women.

Methods

We collected data from participants residing in Jinchang district of Suzhou from January to May, 2010. Serum uric acid, 25-hydroxy vitamin D and other traditional biomarkers including fasting plasma glucose and blood lipids were determined in 1726 women aged above 30 years. Association between vitamin D insufficiency and elevated uric acid was analyzed in premenopausal and postmenopausal women, respectively.

Results

Among postmenopausal women, 25-hydroxy vitamin D level of participants with elevated uric acid was lower than that of those with normal uric acid (median [interquartile range]: 35[28–57] vs 40[32–58], µg/L; P = 0.006). Elevated uric acid was more prevalent in participants with vitamin D insufficiency compared to those without vitamin D insufficiency (16.50% vs 8.08%; P<0.001). Association between vitamin D insufficiency and elevated uric acid was not significant among premenopausal women. However, participants with vitamin D insufficiency were more likely to have elevated uric acid compared with those without vitamin D insufficiency among postmenopausal women (OR, 95% CI: 2.38, 1.47–3.87). Moreover, after excluding individuals with diabetes and/or hypertension, the association of vitamin D insufficiency with elevated uric acid was still significant (OR, 95% CI: 2.48, 1.17–5.44).

Conclusions

Vitamin D insufficiency was significantly associated with elevated uric acid among postmenopausal Chinese Han women. This study suggested that a clinical trial should be conducted to confirm the association of vitamin D insufficiency with hyperuricemia.     

GmSAL1 Hydrolyzes Inositol-1,4,5-Trisphosphate and Regulates Stomatal Closure in Detached Leaves and Ion Compartmentalization in Plant Cells

Inositol polyphosphatases are important regulators since they control the catabolism of phosphoinositol derivatives, which are often signaling molecules for cellular processes. Here we report on the characterization of one of their members in soybean, GmSAL1. In contrast to the substrate specificity of its Arabidopsis homologues (AtSAL1 and AtSAL2), GmSAL1 only hydrolyzes inositol-1,4,5-trisphosphate (IP₃) but not inositol-1,3,4-trisphosphate or inositol-1,4-bisphosphate.The ectopic expression of GmSAL1 in transgenic Arabidopsis thaliana led to a reduction in IP₃ signals, which was inferred from the reduction in the cytoplasmic signals of the in vivo biomarker pleckstrin homology domain–green florescent protein fusion protein and the suppression of abscisic acid-induced stomatal closure. At the cellular level, the ectopic expression of GmSAL1 in transgenic BY-2 cells enhanced vacuolar Na⁺ compartmentalization and therefore could partially alleviate salinity stress.     

数字PCR技术及其在检测领域的应用

随着分子生物学技术的不断发展和需求的多样化,用于核酸检测的各种PCR衍生技术应运而生。数字PCR是一种单分子水平的大规模分区扩增定量核酸检测技术。该技术以微腔室/微孔或微滴作为PCR反应器,无需校准物和绘制标准曲线即可实现对样品初始浓度的绝对定量,具有高灵敏度、高特异性和高精确度的特点。本文详细介绍了数字PCR的技术发展史、作用原理以及仪器平台类型,系统阐述了数字PCR在转基因检测、疾病诊断、环境及食品监管等方面的应用概况,并对该技术的应用前景进行了展望,以期对未来数字PCR的开发利用提供参考。