RNA干扰技术及其在寄生线虫基因功能研究中的应用

介绍了RNA干扰（RNAi）的概念、线虫中RNAi的作用机理、RNAi的三个分子特性，即可遗传性、转移性RNAi的放大效应和RNAi信号的传播特性。从模板dsRNA的获取、dsRNA的导入和线虫中RNAi效应的检测方法三个方面概述了应用RNAi技术研究线虫基因功能的试验方法和最新进展。     

Transport of nutrients and electrolytes across the intestinal wall in pigs

In recent years, intestinal transport processes have been studied in detail regarding both, functional and structural aspects. For monosaccharides different systems have been demonstrated for apical uptake: this includes the high-affinity SGLT1 as a distinct d-glucose system and GLUT5 for fructose. Specifically in pigs a low affinity, high-capacity system for d-glucose and d-mannose with no preference for Na⁺ over K⁺ and a very low affinity system are suggested as further uptake systems. As in other species, basolateral extrusion is mediated by GLUT2. The distributions of monosaccharide transport along the gastrointestinal axis as well as the potential role of paracellular monosaccharide absorption have not yet been clarified.

Amino acids can principally be absorbed by the paracellular and transcellular pathway whereas transcellular transport can either be mediated by facilitated diffusion or secondary active Na⁺-coupled transport. This includes different transport systems for neutral, anionic and cationic acids. In addition, the presence of the di-/tripeptides transport system PEPT1 which depends on an inwardly directed H⁺-gradient has also been confirmed for the pig small intestine, its quantitative proportion is still under debate.

Short chain fatty acids (SCFA) are the major end products of microbial carbohydrate fermentation which occurs along the gastrointestinal tract with the highest production rates in the large intestines. At least two uptake mechanisms have to be assumed, i.e., non-ionic diffusion and anionic exchange via SCFA⁻/HCO₃⁻-exchange. Controversial views still exist to what extent SCFA are metabolized within the epithelial tissue.

Segmental differences between small and large intestines have been demonstrated for Na⁺ absorption. Whereas in the small intestines the major part of Na⁺ absorption is mediated by coupled nutrient transport systems, aldosterone sensitive Na⁺ channels and Na⁺/H⁺-exchange are the dominant mechanisms in the hindgut. For Cl⁻ paracellular transport and anionic Cl⁻/HCO₃⁻-exchange are the major absorptive mechanisms. Cl⁻ secretion is mediated by apical channels which may be activated by toxins of different origin. Different types of Cl⁻ channels have been identified, such as Cystic Fibrosis Transmembrane Regulator (CFTR), Ca-activated Cl⁻ channels (CLCA) and Outwardly Rectifying Cl⁻ Channels (ORCC). Whereas CFTR has clearly been shown for jejunal and colonic epithelial and goblet cells controversy still exists on the relevance of CLCA and ORCC in pigs.

For Ca²⁺ there is evidence that both recently published channels TRPV5 and TRPV6 are also expressed in pig intestinal tissues, however, this has not yet been shown on protein level. From several functional approaches it was demonstrated that phosphate uptake can be mediated by both, a Na⁺-dependent transcellular component and paracellularly. On a molecular basis it is uncertain whether the transport protein of transcellular mechanism belongs to the NaPi-IIb cotransporter family.     

三倍体梨新品种‘龙园洋红’

 龙园洋红’是三倍体梨新品种, 属短枝型, 亲本为56-5-20和乔玛, 抗寒力强, 平均单果质量185.5 g, 最大可达650.6 g, 果实为不规则长椭圆形, 果型指数1.13。果皮底色黄, 有红晕。果肉白色,细软多汁, 可溶性固形物16.05% , 可溶性糖11.68%。可滴定酸0.81% , 果实9月15日左右成熟, 风味酸甜适度、有香味。     

牛GDF9和BMP15基因遗传变异与双胎性状的关系研究

以生长分化因子9(Growth differentiation factor 9,GDF9)基因和骨形态发生蛋白15(Bone morphogenet-ic protein 15,BMP15)基因作为牛双胎性状的候选基因,研究了它们在鲁西牛、秦川牛、南阳牛和中国荷斯坦牛4个品种中的遗传变异,并在鲁西牛群体中研究了其多态位点与双胎性状的关系。结果表明:在鲁西牛中GDF9基因的3′UTR发现缺失突变,而其它3个品种中没有发现该突变。对鲁西牛群体中该多态位点与单、双胎性状之间进行卡方显著性检验表明,单胎牛群体与双胎牛群体基因型分布有极显著的差异(P=0.006),双胎牛群体的B等位基因频率明显大于单胎牛群体。通过生物信息学分析表明,突变体mRNA的二级结构与野生型相比总自由能值差异不大,但突变体mRNA翻译起始位点的二级结构稳定性明显大于野生型。在鲁西牛、南阳牛和秦川牛的BMP15基因中发现编码区第759~762位有GAAA 4个碱基存在缺失突变,但没有检测到突变纯合个体,中国荷斯坦牛中没有检测到该突变。卡方显著性检验表明单胎牛群体和双胎牛群体在该位点基因型组成差异不显著(P=0.947)。     

用荧光定量RT-PCR方法检测猪瘟病毒

为了建立能特异检测不同基因型猪瘟病毒(Classical swine fever virus,CSFV),同时又能区分其他瘟病毒的基因检测方法,本实验针对CSFV基因组5′端非编码区设计并合成了简并引物和TaqMan探针,在优化反应条件的基础上,成功地建立了特异检测CSFV的荧光定量RT-PCR检测方法。再以已知滴度的CSFV石门株血毒总RNA反转录产物建立标准品,该标准品可以用于定量临床样品中的CSFV滴度,所建立的荧光定量PCR方法可以灵敏地检测出10~(-0.82)个TCID_(50)病毒含量。最后用建立的方法对108份临床样品进行检测并同时进行病毒分离,荧光定量PCR方法检测出73份阳性样品且与病毒分离的符合率为100%,而常规RT-PCR只检测出54份阳性样品,表明本荧光定量RT-PCR法在检测猪瘟病料上具有潜在的应用价值。     

Comparison of S-RNase,RNase T1, T2, and A effects on growth inhibition and RNA degradation of in vitro-cultured pear pollen

To obtain the basic information on fruit set regulation, effects of several RNases including S-RNase on pollen tube growth and RNA degradation in the tube were studied in the pear. Purified S-RNase from the Japanese pear ‘Kosui’ (S₄S₅) predominantly inhibited the growth of ‘Kosui’ pollen tubes (self) in vitro at 0.28 unit μL⁻¹, but it inhibited ‘Chojuro’ (S₂S₃) pollen (cross) only slightly. The same unit of RNase T₁ (EC 3.1.27.3) clearly inhibited the pollen tube growth, but the action was significantly weaker than that of the S-RNase against the self-pollen. Inhibitory effect of RNase T₂ (EC 3.1.27.1) and RNase A (EC 3.1.27.5) was only slight. The proteins other than the S-RNase extracted from pear style did not have any inhibitory action, though they possessed RNase activity 3.8 times higher than S-RNase. Thus, RNases tested here could not substitute for the S-RNase in specific inhibition against the self-pollen tube growth. Total RNA degradation by each RNase occurred in the pollen tubes as following order; S-RNase (self) ≥T₁ > T₂ ≥ A > S-RNase (cross). Degradation degree of 28S and 18S rRNA was as follows; S-RNase (self) > A > T₁ > T₂ > S-RNase (cross). The degradation of 5.8S and 5S rRNA was; S-RNase (self) > S-RNase (cross) > A > T₂ > T_1. The degree of rRNA degradation was, thus, not always in parallel with the degree of pollen growth inhibition. The S-RNase may degrade not only rRNA but also mRNA essential for pollen tube growth, and may be specifically adapted to inhibit the growth of self-pollen tubes. Therefore, controlling S-RNase amount in the style will produce self-thinning cultivars efficiently, which are unnecessary not only for hand-pollination but fruit-thinning practices in the pear. Practically, cultivar with weak self-incompatibility and small amount of S-RNase, such as ‘Okusankichi’, may be an expecting candidate for breeding self-thinning cultivars.     

Construction and identification of eukaryotic expression vector of bcl-2 siRNA

AIM: To construct eukaryotic expression vector of small interfering RNA(siRNA) specific to bcl-2 and investigate the effect of recombinant plasmid on suppressing bladder cancer cell growth.METHODS: siRNA of bcl-2 gene was designed according to the principle of RNAi-based medicine, and was converted into cDNA coding expression of small hairpin RNAs(shRNA) of siRNA. The cDNA was synthesized and inserted into plasmid pGenesil-1. The recombinant eukaryotic expression vectors of pGenesil-1545 and pGenesil-1555 were controlled by the U6 promoter of RNA polymerase Ⅲ, identified by the restriction map and the sequence analysis, and transfected into T24 cells. After T24 cells were transfected for 72 h, expression of bcl-2 mRNA was assayed by RT-PCR; and MTT was used to observe the proliferation of T24 cells.RESULTS: The recombinant plasmids of pGenesil-1545 and pGenesil-1555 were identified by the restriction map and the sequence analysis. The sequences completely coincided with the designs. The expression of the bcl-2 mRNA in T24 cells transfected with recombinant plasmid decreased nearly 80%, and the growth of T24 cells was suppressed significantly.CONCLUSION: The siRNA eukaryotic expression vector against bcl-2 gene is successfully constructed. It effectively downregulates the expression of bcl-2 in T24 cells and suppresses the cell growth.     

RNAi技术在转基因西瓜抗病毒病上的研究与应用

对RNAi技术的概念、研究进展及西瓜病毒病的危害及其发生特点、RNAi技术在西瓜抗病毒病上的研究进展进行了阐述,并对存在的一些问题作了简要探讨与展望。     

靶向S基因外源性microRNA抑制TGEV增殖效果的研究

应用生物信息学软件对猪传染性胃肠炎病毒(TGEV)S基因进行分析,筛选出可能与S基因有相互作用的外源miRNA:amiRNA-S-28035,amiRNA-S-28038,amiRNA-S-28165。之后,利用脂质体将构建好的相应的表达载体瞬时转染至PK-15细胞;通过Q-PCR和间接免疫荧光方法检测其对S基因的抑制作用;CPE分析和TCID50测定检测其对TGEV增殖的抑制效果。结果发现,3个外源性miRNA均降低了S基因mRNA的转录和蛋白的表达,其中amiRNA-S-28038对TGEV mRNA的平均抑制率可达64.6%,最高可达69.9%,表明外源性microRNA可以通过靶向TGEV基因组来抑制TGEV的复制。研究结果为猪传染性胃肠炎的预防和治疗提供了新的思路。     

小麦小分子RNA TaMIR1129介导植株抵御低氮逆境功能研究

本研究针对迄今有关小麦小分子RNA(miRNA)家族成员介导植株氮素吸收和利用机理尚少见报道的现状,对TaMIR1129的表达特征和介导植株抵御低氮逆境功能进行了研究。结果表明,TaMIR1129呈低氮胁迫诱导表达,表现为随氮浓度降低(0.02~6mmol/L)和处理时间延长(0~48h)表达水平不断增高特征。此外,低氮诱导的高表达水平在恢复供氮后表达下调。表明该miRNA对介质中氮素应答呈典型的时间及浓度依赖特征。TaMIR1129作用2个靶基因,包括Molybdenum cofactor sulfurase(TaMCS)和Major facilitator family transporter(TaMFFT),上述基因应答低氮特征与TaMIR1129相反。遗传转化结果表明,超表达TaMIR1129具有显著增强植株抵御低氮逆境的能力。表现为与野生型对照相比,转基因系Sen 1和Sen 2低氮处理后植株形态增大,干质量增加,氮累积量增多。表明TaMIR1129与作用靶基因构建miRNA/target模块在介导植株抵御低氮逆境中发挥重要作用。