葡萄果实cDNA文库的构建及鉴定

构建葡萄果实cDNA文库是研究果实发育相关基因表达调控的基础工作.本实验从巨峰葡萄果实中提取了高质量的RNA,利用MMLV反转录酶把总RNA中的mRNA反转录成cDNA第一链,用特异引物进行LD-PCR扩增合成双链cDNA.将分级纯化的带有粘性末端双链cDNA与λTrip Ⅰ Ex2载体连接,重组载体体外包埋成为cDNA原始文库.初步鉴定原始文库的滴度为1.32×106 pfu/mL,文库扩增后滴度达1.45×1010 pfu/mL.从扩增文库中随机挑取250个克隆进行PCR鉴定,结果表明重组率为98.7%,插入片段大小在0.5～2.0 kb之间,因此,本研究成功构建了葡萄果实cDNA文库.     

基于18S rRNA的鸡组织滴虫病PCR诊断方法的建立

河南某鸡场约4周龄鸡疑似发生鸡组织滴虫病.根据鸡组织滴虫18 S rRNA序列设计引物,提取肝脏、盲肠内客物寄生虫DNA,采用PCR方法检测.结果表明,PCR扩增到与预期大小一致的产物,经测序比对,证实为鸡组织滴虫感染.所建立的PCR方法具有灵敏、特异、快速等优点,不仅能用于鸡组织滴虫病临床诊断,还能用于开展流行病学研...     

条锈菌侵染早期小麦叶片RNA和rRNA的合成

 采用示踪方法研究了条锈菌侵染早期小麦叶片的RNA和rRNA合成。结果表21h期间,表现不亲和性反应的叶片总RNA合成有所增加,而亲和性反应寄主叶片则低rRNA合成未受侵染影响。在接种后39~45h期间,只有呈亲和性反应的寄主叶片RNA和大幅度增加。对rRNA各组分合成的测定表明条锈菌侵染所影响的只是大分子量rRNA,胞质和叶绿体rRNA的合成也存在差别。文中讨论了上述变化的病理学意义。     

Molecular Variability of the Capsid Protein of the Prune Dwarf Virus

Sequences of the capsid protein gene and the preceding intergenic region of eleven isolates of prune dwarf virus from central Europe were determined. The isolates were obtained from plum, cherry and peach trees. Comparison of all sequenced isolates (including two sequences published previously) revealed high (88%) conservation of the capsid protein gene. The highest degree of identity was observed in the C-terminal half, where only 13 amino acid substitutions could be observed in contrast to the N-terminal half with 22 substitutions. No reasonable correlation between amino acid substitutions and host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other ilarviruses revealed apple mosaic virus, elm mottle virus, lilac ring mottle virus and prunus necrotic ringspot virus as the most related to prune dwarf virus. Unlike the isolates of related prunus necrotic ringspot virus all the isolates of prune dwarf virus shared extensive conservation of the intergenic region. Portions of RNA3 were selected for design of universal primers for PCR detection.     

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。     

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。