哺乳动物中RNA干扰研究进展

RNA干扰(RNAi)由双链RNA引起,广泛存在于动、植物中的序列特异性转录后基因沉默,是生物体在进化过程中,抵御病毒感染及由于重复序列和突变引起的基因组不稳定性的保护机制。论文从RNAi现象、RNAi发生机制、哺乳动物中的RNAi现象和RNAi与抗病毒感染等几个方面做了综述,详细介绍RNAi在哺乳动物中的应用。     

Effect of shRNA-mediated survivin gene silencing on sensitivity of lymphoma cells to adriamycin

AIM: This study was designed to use RNA interference technique to down-regulate the expression of survivin gene in human Burkitts lymphoma cell line Daudi and to explore the effect on sensitivity of Daudi cells to adriamycin. METHODS: The survivin-shRNA expression vector was constructed and transfected into Daudi cells. Expression of survivin mRNA and protein were assessed by RT-PCR and Western blotting analysis, respectively. Apoptosis index of transfected Daudi cells was quantified by flow cytometry. The sensitivity of Daudi cells to adriamycin (ADR) before and after transfection was detected by MTT test. RESULTS: The mRNA and protein levels of survivin were down-regulated by 62.32% and 61.88%, respectively, compared to those in control-shRNA treated group and PBS treated group (P<0.05). Meanwhile, the apoptosis index was significantly increased (19.10%±2.15%), compared to that in control group (4.48%±1.54%) and PBS group (4.35%±1.37%, P<0.05). The 50% inhibition concentration (IC50) of ADM to Daudi cells was significantly decreased (0.25±0.43) μmol/L, compared to that in control group (0.87±0.21) μmol/L and PBS group (0.91±0.36) μmol/L, P<0.05. CONCLUSION: Down-regulation of survivin expression in Daudi cells by shRNA effectively induces apoptosis and increases the sensitivity of Daudi cells to ADR.     

Heritability of, and genetic correlations among, forage and seed yield traits in Ladino white clover

Sixteen white clover genotypes and their half-sib progenies were grown in pure stand. Each clover progeny was also grown in a mixed stand that also included three grass varieties of different species. Dry matter (DM) yield was measured over 2 years and seed yield at the second year. Competitive ability of clover families was defined as the ratio between mixed stand and pure stand for clover DM yield. Narrow-sense heritability from parent-offspring regression of pure stand data was high to moderate (h²0.50) for DM yield, seed yield and most of their components. Persistence as predicted by stolon density showed negative genetic correlations with seed yield (r_g= -0.70) and DM yield (r₂= -0.60), whereas seed yield and DM yield tended towards a negative correlation (r₂=0.45). Sizes of different vegetative or reproductive organs were generally correlated positively. Evidence was provided that the set of parent genotypes represents well the genetic variation available within the Ladino gene pool. The results highlighted the difficulty of combining relatively short-term DM yield, persistence and seed yield into a unique plant type. Differences in competitive ability emerged only in the second year, better ability being related mainly to petiole length among the traits assessed in pure stand.     

稗草影响大豆根瘤固氮的机制研究之一——水分干扰机制

在全自控人工气候室内系统研究了稗草对大豆根瘤固氮的水分干扰机制。稗草在每盆3株的密度下使播后53-63淹水期间及淹水期后1天的大豆单株根瘤固氮速率平均分别比无草对照提高了16.6%和30.0%,在每盆10株的密度下分别使其比无草对照提高了34.2%和67.5%。在不淹水的情况下播后30-70天,稗草在每盆3株和10株的密度下使其所在处理的平均日蒸发蒸腾量分别比无草对照增加了23.8%和50%,削弱了水分对大豆植株的供应水平;而土壤水分供应水平从其田间持水量的80%降低到35%时,大豆根瘤的氧气传导速率从0.38cm/s下降到0.20cm/s,净光合速率从18.81mg/dm#+2/h降低到4.04mg/dm#+2/h,每盆10株稗草对大豆单株结瘤量及根瘤固氮酶活性的抑制率则分别从8.2%和17.8%升高到18.3%和19.6%,其对大豆单株根瘤固氮速率的抑制率也随之从21.5%上升到33.1%。证明通过干扰土壤水分供应水平,首先影响大豆植株的同化物产量及根瘤内的氧气供应,进而影响大豆植株的根瘤形成和根瘤固 氮酶的固氮活性,是稗草影响大豆根瘤固氮的主要机制之一。     

靶向cofilin2基因RNAi对鸭病毒性肠炎病毒增殖的影响

旨在探讨cofilin2基因的表达在鸭病毒性肠炎病毒(DEV)增殖过程中的影响.本研究通过已获得的鸭源cofilin2全基因序列(GenBank登录号:MF434775),设计出4对shRNA序列,插入载体中,并转染到鸭胚成纤维细胞中,经FQ-PCR方法和荧光显微镜观察筛选出具有较高干扰效率的shRNA序列,用干扰效率...     

RNA干扰对大鲵蛙病毒主要功能基因表达及其增殖的影响

采用q PCR与病毒滴度测定观察RNA干扰(RNA interference,RNAi)对大鲵蛙病毒(Chinese giant salamander ranavirus,CGSRV)主要衣壳蛋白(major capsid protein,MCP)、甲基转移酶(methyltransferases,MTases)、DNA多聚酶(DNA polymerase)基因表达与病毒增殖的影响。结果表明,小干扰RNA(small interfering RNA,siRNA)能推迟鲤鱼上皮瘤细胞(epithelioma papillosum cyprini,EPC)出现病变,且病变程度也较对照组轻。在各功能基因表达量上,NC-FAM对照组与阴性对照组差异不显著(P0.05);而干扰组的干扰率极显著高于阴性对照组,其中siR-DP-1组siRNA对MCP基因的干扰率为79%,极显著高于其余组(P0.01);siR-MT-1、siR-MT-2组siRNA对MTases基因的干扰率为77%,极显著高于其余组(P0.01);siR-DP-1组siRNA对DNA polymerase基因的干扰率为79%,极显著高于其余组(P0.01)。干扰实验组病毒滴度与NC-FAM对照组和阴性对照组相比也有不同程度的降低。阴性对照组lg TCID50为8.362,NC-FAM对照组lg TCID50为7.848,siR-MCP、siR-MT、siR-DP实验组最低lg TCID50分别为5.764、5.317、5.362。证实RNAi能够抑制CGSRV主要功能基因的表达并影响病毒的复制。     

RNA干扰技术抑制新家坡石斑鱼虹彩病毒感染细胞多肽ICP46与绿色荧光蛋白融合基因的表达

新加坡石斑鱼虹彩病毒（singapore grouper iridovirus,SGIV）是一种严重的能引起全身性疾病的病原体,对石斑鱼养殖造成了重大的经济损失。将含有SGIV感染细胞多肽ICP46（infected cell polypeptides 46）基因的真核表达载体pEGFP-ICP46转染到胖头鲤细胞（fathead minnow cells,FHM）中进行融合表达,用荧光显微镜观察到ICP46-GFP融合蛋白主要分布于FHM细胞的细胞质中。根据SGIV-ICP46的序列,设计并体外化学合成了特异性干扰SGIV-ICP46的siRNA（siRNA-ICP46）,与pEGFP-ICP46共转染到FHM细胞中,通过荧光显微镜观察不同时间点荧光强度的变化。转染后24~48 h,实验细胞（共转染siRNA-ICP46和pEGFP-ICP46）和阳性对照细胞（共转染siRNA-GFP和pEGFP-ICP46）中的荧光微弱,发荧光的细胞数量较阴性对照（共转染siRNA-Negative和pEGFP-ICP46）少70%左右,但其后实验细胞和阳性对照细胞的荧光强度开始增强,在转染后72 h其与阴性对照组已差别不大。说明体外化学合成的siRNA-ICP46转染后24~48 h可有效抑制FHM细胞中外源导入SGIV-ICP46基因的表达。     

Breeding for provitamin A biofortification of maize (Zea mays L.)

Vitamin A deficiency is widely prevailing in children and women of developing countries. Deficiency of vitamin A causes night blindness, growth retardation, xerophthalmia and increases the susceptibility against epidemic diseases. Among different interventions of overcoming malnutrition, biofortification is the most acceptable and preferred intervention among researchers, growers and consumers. Maize is grown and consumed in those regions where vitamin A deficiency is most prevalent; thus, targeting this crop for provitamin A biofortification is the most appropriate solution. Different breeding strategies including diversity analysis, introduction and stability analysis of exotic germplasm, hybridization, heterosis breeding, mutagenesis and marker‐assisted selection are practised for exploring maize germplasm and development of provitamin A‐enriched cultivars. Genome‐wide association selection and development of transgenic maize genotypes are also being practised, whereas RNA interference and genome editing tools could also be used as potential strategies for provitamin A biofortification of maize genotypes. The use of these breeding strategies for provitamin A biofortification of maize is comprehensively reviewed to provide a working outline for maize breeders.     

浑水膜孔灌多向交汇入渗及减渗特性

浑水灌溉是我国黄河流域引黄灌区灌溉的主要特点,其中浑水中的泥沙含量对土壤入渗特性有较大的影响,为揭示浑水膜孔灌交汇入渗的减渗特性,通过开展浑水及清水膜孔灌入渗试验,建立了浑水膜孔灌自由入渗相对于清水的减渗率及浑水膜孔灌多向交汇入渗相对于自由入渗的减渗率与入渗时间之间的量化关系,提出了由清水膜孔灌自由入渗量推求浑水膜孔灌多向交汇入渗量的模型。结果表明:浑水膜孔灌自由入渗较清水的减渗量随入渗时间的延长而增大,但增大幅度慢慢变小,入渗后期其减渗量基本呈线性增加,其减渗率随时间的增大先快速增大后缓慢减小,入渗后期减渗率基本稳定;当入渗产生交汇后,入渗能力明显减小,多向交汇入渗和单向交汇入渗相对自由入渗的减渗率均随入渗时间的增长而增大,多向交汇入渗相对单向交汇入渗也存在减渗作用,3条减渗率曲线的变化率随着时间的增长而逐渐减小。