A phylogenetic study of <i>Drosophila</i>splicing assembly chaperone RNP-4F associated U4-/U6-snRNA secondary structure

The rnp-4f gene in Drosophila melanogaster encodes nuclear protein RNP-4F. This encoded protein is represented by homologs in other eukaryotic species, where it has been shown to function as an intron splicing assembly factor. Here, RNP-4F is believed to initially bind to a recognition sequence on U6-snRNA, serving as a chaperone to facilitate its association with U4-snRNA by intermolecular hydrogen bonding. RNA conformations are a key factor in spliceosome function, so that elucidation of changing secondary structures for interacting snRNAs is a subject of considerable interest and importance. Among the five snRNAs which participate in removal of spliceosomal introns, there is a growing consensus that U6-snRNA is the most structurally dynamic and may constitute the catalytic core. Previous studies by others have generated potential secondary structures for free U4-and U6-snRNAs, including the Y-shaped U4-/U6-snRNA model. These models were based on study of RNAs from relatively few species, and the popular Y-shaped model remains to be systematically re-examined with reference to the many new sequences generated by recent genomic sequencing projects. We have utilized a comparative phylogenetic approach on 60 diverse eukaryotic species, which resulted in a revised and improved U4-/U6-snRNA secondary structure. This general model is supported by observation of abundant compensatory base mutations in every stem, and incorporates more of the nucleotides into base-paired associations than in previous models, thus being more energetically stable. We have extensively sampled the eukaryotic phylogenetic tree to its deepest roots, but did not find genes potentially encoding either U4-or U6-snRNA in the Giardia and Trichomonas data-bases. Our results support the hypothesis that nuclear introns in these most deeply rooted eukaryotes may represent evolutionary intermediates, sharing characteristics of both group II and spliceosomal introns. An unexpected result of this study was discovery of a potential competitive binding site for Drosophila splicing assembly factor RNP-4Fto a5’-UTR regulatory region within its own pre-mRNA, which may play a role in negative feedback control.

禽4型副黏病毒F基因的克隆及原核表达载体构建

扩增禽4型副黏病毒(Avian paramyxovirus type 4,APMV-4)新疆野鸭源分离株APMV-4/Pintail/CH(XJ)/01/2016的F基因,并构建原核表达载体。参考GenBank上公布的APMV-4序列,通过DNAStar设计F基因特异性引物,以APMV-4/Pintail/CH(XJ)/01/2016基因组RNA为模板,采用RT-PCR方法扩增F基因编码区,连接于pMD19-T载体,用BamHⅠ和HindⅢ酶切原核表达载体pET-30a重组质粒,将酶切产物克隆至pET-30a多克隆位点上,转化至大肠杆菌DH5α感受态细胞中,构建F基因原核表达载体,并对阳性重组质粒进行PCR和测序鉴定。结果显示,扩增出的片段大小为1701 bp,经测序鉴定该扩增片段为F基因全长,与GenBank中收录的APMV-4序列核苷酸相似性为99%;连接表达载体结果表明,原核表达载体pET-30a-F成功构建,为APMV-4诊断方法的建立提供了良好的技术基础。

Regulation of Expression for the RNP-4F Splicing Assembly Factor in the Fruit-Fly <i>Drosophila melanogaster</i>

Intron splicing in eukaryotic organisms requires the interactions of five snRNAs and numerous different proteins in the spliceosome. Although the molecular mechanism behind splicing has been well studied, relatively little is known about regulation of expression for these splicing factor proteins. One of these proteins is the evolutionarily-conserved Drosophila RNP-4F splicing assembly factor. This protein is transcribed from a single gene into two developmentally regulated mRNAs that differ in their 5’-UTR structure. In the longer isoform, known to be abundant in the developing fly central nervous system, a conserved retained intron which folds into a stem-loop has been implicated in expression control of the mRNA. Here, we describe construction and utilization of several new rnp-4f gene expression study vectors using a GFP reporter in the ΦC31 system. The results confirm our previous observation that presence of the regulatory stem-loop enhances RNP-4F protein expression. However, in that study, the enhancement factor protein was not identified. We show here that overexpression of the RNP-4F transgene compared to the control results in additional translation, as indicated by the GFP reporter in the fluorescent images. These results are interpreted to show that RNP-4F protein acts back on its own mRNA 5’-UTR regulatory region via a feedback pathway to enhance protein synthesis in the developing fly central nervous system. A model is proposed to explain the molecular mechanism behind rnp-4f gene expression control.

DNA methylation patterns of banana leaves in response to Fusarium oxysporum f. sp. cubense tropical race 4

Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense tropical race 4 （Foc TR4）, is a serious soil-borne fungal disease. Now, the epigenetic molecular pathogenic basis is elusive. In this study, with methylation-sensitive amplification polymorphism （MSAP） technique, DNA methylation was compared between the leaves inoculated with Foc TR4 and the mock-inoculated leaves at different pathogenic stages. With 25 pairs of primers, 1 144 and 1 255 fragments were amplified from the infected and mock-inoculated leaves, respectively. DNA methylation was both changed and the average methylated CCGG sequences were 34.81 and 29.26% for the infected and the mock-inoculated leaves. And DNA hypermethylation and hypomethylation were induced by pathogen infection during all pathogenic stages. Further, 69 polymorphic fragments were sequenced and 29 of them showed sequence similarity to genes with known functions. And RT-PCR results of four genes indicated that their expression patterns were consistent with their methylation patterns. Our results suggest that DNA methylation plays important roles in pathogenic response to Foc TR4 for banana.

A molecular, phylogenetic and functional study of the <i>dADAR</i>mRNA truncated isoform during <i>Drosophila</i>embryonic development reveals an editing-independent function

Adenosine Deaminases Acting on RNA (ADARs) have been studied in many animal phyla, where they have been shown to deaminate specific adenosines into inosines in duplex mRNA regions. In Drosophila, two isoform classes are encoded, designated full-length (contains the editase domain) and truncated (lacks this domain). Much is known about the full-length isoform, which plays a major role in regulating functions of voltage-gated ion channel proteins in the adult brain. In contrast, almost nothing is known about the functional significance of the truncated isoform. In situ hybridization shows that both isoform mRNA classes are maternally derived and transcripts for both localize primarily to the developing central nervous system. Quantitative RT-PCR shows that about 35% of all dADAR mRNA transcripts belong to the truncated class in embryos. 3’-RACE results show that abundance of the truncated isoform class is developmentally regulated, with a longer transcript appearing after the mid-blastula transition.3’-UTR sequences for the truncated isoform have been determined from diverse Drosophila species and important regulatory regions including stop codons have been mapped. Western analysis shows that both mRNA isoform classes are translated into protein during embryonic development, as full-length variant levels gradually diminish. The truncated protein isoform is present in every Drosophila species studied, extending over a period spanning about 40 x 106 years, implying a conserved function. Previous work has shown that a dADAR protein isoform binds to the evolutionarily conserved rnp-4f pre-mRNA stem-loop located in the 5’-UTR to regulate splicing, while no RNA editing was observed, suggesting the hypothesis that it is the non-catalytic truncated isoform which regulates splicing. To test this hypothesis, we have utilized RNAi technology, the results of which support the hypothesis. These results demonstrate a novel, non-catalytic function for the truncated dADAR protein isoform in Drosophila embryonic development, which is very likely evolutionarily conserved.

香蕉枯萎病菌4号生理小种农杆菌介导遗传转化体系的建立及T-DNA插入突变体的筛选

【目的】通过农杆菌介导的遗传转化（ATMT）方法，建立香蕉枯萎病4号生理小种的高效转化体系，构建病原菌的突变体库并进行筛选，为已突变基因提供分子标记，在分子水平上对香蕉枯萎病菌的功能基因进行深入研究。【方法】通过单因子变量（试验材料、真菌孢子浓度、农杆菌预诱导终浓度、IM诱导培养基pH、共培养介质、共培养时AS浓度、共培养温度）试验，研究影响农杆菌介导的遗传转化效率的关键因素，并通过形态观察与致病性试验筛选突变体。【结果】得到的最佳转化条件为：农杆菌预诱导浓度OD600=0.8，病原菌孢子浓度106 CFU/mL，转化受体材料为分生孢子，共培养培养基pH 5.4，共培养温度25 ℃，共培养培养基中AS浓度200 μmol/L，共培养介质为硝酸纤维素膜。通过条件优化，转化效率可达到800-900个转化子/106个孢子。【结论】通过农杆菌介导的遗传转化成功将GFP基因转入病原菌中并表达，构建了病原菌的T-DNA插入突变体库，并通过筛选得到多个表型和致病性发生变化的突变体，为香蕉枯萎病菌基因组功能注释的研究奠定了基础。

中国小麦条锈病菌鉴别寄主中4抗病基因遗传组成分析

中4是中国小麦条锈菌生理小种的重要鉴别寄主之一。采用常规杂交遗传分析法和花粉母细胞染色体镜检,明确中4抗条锈病基因遗传组成,并探讨利用中国春ph1b突变体分析小麦近缘属(种)抗条锈病基因。将中4分别与中国春ph1b突变体和感病品种铭贤169杂交,对亲本及其杂交后代进行苗期抗条锈性鉴定和遗传分析,发现中4对条锈菌小种CY31和CY32的抗病性由1对显性基因控制;通过等位性分析和抗谱比较,发现中4对小种CY32的抗病基因与T. spelta album、Moro及K733中的抗条锈病基因不同,对中国春ph1b突变体×中4组合的F2代植株染色体数目及其核型变化的研究表明,F2代单株的抗条锈性与来自中间偃麦草X组染色体增加有关,并导致F2单株染色体数目发生变化,且X组染色体在F2代群体对小种CY31表现为抗病和感病植株中随机分布。