植物中miR160/miR167/miR390家族及其靶基因研究进展

microRNA(miRNA)是真核生物中广泛存在的一类长21～23个核苷酸的非编码RNA分子,通过与靶基因mRNA的特异结合调节基因转录后表达,在调控细胞周期、生物体发育时序等方面起重要作用。miR160、miR167和miR390 3个miRNAs家族均为靶向ARF(Auxin Response Factor)基因家族,预示着它们在行使调控功能的过程中既有相似性又有特异性。笔者详细阐述了miR160/miR167/miR390在植物发育过程中的调控作用及与其靶基因之间的相互调控关系,发现miR160/miR167/miR390均在植物生长发育过程中发挥重要的作用,但miR160则侧重于调控胚胎和根的发育,miR167侧重于调控植物花和果实的发育,而miR390则对植物横向器官的发育具有调控作用,并且,miR160/miR167/miR390与其靶基因之间存在反馈调节作用。     

敲除和过表达miRNA446以研究其靶基因APP1的表达模式

MicroRNA是一类小的非编码RNA,参与植物生长发育和胁迫应答等多种生理过程。在本课题组前期研究中,通过基因芯片数据发现了一系列在胁迫应答中表达量有变化的microRNA。其中以miRNA446的变化尤为显著。为进一步探求miRNA446在胁迫应答中的变化与功能,本实验通过在水稻中分别敲除和过表达miR446,检测干旱和冷胁迫下,miR446及其靶基因APP1的表达模式。结果表明,过表达株系中,miR446的表达量与野生型的持续升高不同,是先降后升的,而APP1基因由于受到高水平miR446的剪切作用而一直降低。敲除株系中,miR446的表达量被完全抑制,在整个胁迫过程中均保持接近于零的水平,而APP1在胁迫中也仅有微弱的先降后升趋势。     

Delayed resistance to Cucumber mosaic virus mediated by 3’UTR-derived hairpin RNA

RNA silencing has been shown to function in the plant antivirus defense response, leading to viral RNA degradation induced by vsiRNA-containing RISC cleavage activity. Cucumber mosaic virus （CMV） 3′UTR sequences share a high conservation of nucleotide sequence and secondary structures that are important for CMV replication. Here, in an attempt to simultaneously target the multiple genomic and subgenomic RNAs of CMV for degradation, CMV 3′UTR were used to design hairpin RNA （hpRNA） to transform tobacco （Xanthi. nc） so as to constitutively produce viral siRNAs. Most of the transgenic plants expressing CMV Q strain （Q-CMV, subgroup Ⅱ strain） RNA3 3′UTR-derived hpRNA showed delayed resistance to Q-CMV infection and exhibited recovery phenotypes. Compared with Q-CMV-inoculated leaves, the upper leaves showed weak or no disease symptoms and a reduced accumulation level of viral RNAs. Together with transient assays, our results indicate that the 3′UTR-derived siRNAs were biologically active in targeting viral RNA for degradation. Recovery resistance in transgenic plants was also observed against subgroup IB strain SD-CMV infection, indicating a broad-spectrum anti-CMV effect of the 3′UTR-based antiviral silencing. Northern blot assays indicated that there was no strong correlation between the degree of resistance and the accumulation level of 3′UTR-derived siRNAs, suggesting that to target a highly structured RNA, such as the CMV 3′UTR, the quantity of siRNAs may not be the only determinant of silencing efficiency. Target RNA secondary structures may also affect target accessibility, siRNA-containing RISC-target recognition and the consequent antiviral effect.     

Do rice water weevils and rice stem borers compete when sharing a host plant？

The rice water weevil （RWW） Lissorhoptrus oryzophilus Kuschel （Coleoptera： Curculionidae） is an invasive insect pest office Oryza sativa L. in China. Little is known about the interactions of this weevil with indigenous herbivores. In the present study, adult feeding and population density of the weevil, injury level of striped stem borer Chilo suppressalis （Walker） （Lepidoptera： Pyralidae） and pink stem borer Sesamia inferens （Walker） （Lepidoptera： Noctuidae） to flee, as well as growth status of their host plants were surveyed in a rice field located in Southeastern Zhejiang, China, in 2004 with the objective to discover interspecific interactions on the flee. At tillefing stage, both adult feeding of the weevil and injury of the stem borers tended to occur on larger tillers （bearing 5 leaves） compared with small tillers （bearing 2-4 leaves）, but the insects showed no evident competition with each other. At booting stage, the stem borers caused more withering/dead hearts and the weevil reached a higher density on the plants which had more productive tillers and larger root system; the number of weevils per tiller correlated negatively with the percentage of withering/dead hearts of plants in a hill. These observations indicate that interspecific interactions exist between the rice water weevil and the rice stem borers with negative relations occurring at booting or earlier developmental stages of rice.     

Ammonium improves iron nutrition by decreasing leaf apoplastic pH of sunflower plants (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L. cv. Frankasol)

The effect of nitrogen form on pH and concentration of soluble iron (Fe) in leaf apoplast was investigated in hydrophonically grown sunflower plants (Helianthus annuus L. cv. Frankasol), and the mechanism underlying the improved Fe nutrition by ammonium (NH₄) supply was also elucidated. Ammonium supply ameliorated Fe nutrition of plants grown without Fe through decreasing apoplastic pH and increasing soluble Fe concentration in apoplastic fluid of young leaves. The soluble Fe concentration in apoplastic fluid and cell sap of young leaves, and xylem exudates of NH₄ fed-plants was higher than that of nitrate (NO₃) fed-plants, and no typical Fe-deficiency chlorosis in young leaves was observed in NH₄, fed plant without Fe supply. The apoplastic pH was 6.15 and 5.94 in young leaves of Fe-deficient plants fed respectively with NO₃ and NH₄, while in Fe-sufficient plants, the apoplastic pH was 6.43 with NO₃, and 5.50 with NH₄ supply. In primary leaves, the apoplastic pH was around 6.25 irrespective of nitrogen form and Fe supply. The pH of xylem exudate was 5.72 in Fe-deficient plants fed with NO₃ and 5.49 with NH₄. Iron nutrition increased the pH of xylem exudate by 0.27 and 0.16 unit under NO₃ and NH₄ supply respectively.     

基于生物信息学构建前列腺癌单细胞层面lncRNA-miRNA-mRNA调控网络

利用生物信息学筛选差异表达基因（Differentially Expressed Genes,DEGs）,构建前列腺癌（Prostate Cancer,PCa）单细胞层面lncRNA-miRNA-mRNA调控网络,在分子水平上研究前列腺癌的发生发展过程及预后指标。首先从基因表达综合数据库（GEO）和癌症基因组图谱（TCGA）数据库分别下载单细胞RNA测序数据GSE157703和转录组测序数据TCGA-PRAD,通过R语言筛选差异表达信使RNA （DEmRNA）和差异表达长非编码RNA （DElncRNA）,利用相关软件预测并构建lncRNA-miRNA-mRNA调控网络;然后使用R语言进行基因本体论（Gene Ontology,GO）和京都基因与基因百科全书（Kyoto Encyclopedia of Genes and Genomes,KEGG）分析,运用STRING数据库、Cytoscape软件建立蛋白相互作用网络,同时对单细胞RNA测序数据进行质控、降维、聚类和分群,构建单细胞层面的lncRNA-miRNA-mRNA调控网络;最后进行生存分析,通过人类蛋白质图谱进行验证。结果显示,共得到3 013个DEmRNA和1 101个DElncRNA,筛选出51个lncRNA、27个miRNA和97个mRNA构建lncRNA-miRNA-mRNA调控网络。GO分析揭示靶基因在腺状细胞迁移、化学突触传递的调节等生物学过程富集,KEGG分析揭示miRNA在癌症、轴突导向和胞间的黏附等信号通路富集。通过PPI得到Top 10基因,据此整合单细胞水平上PCa单细胞转录组测序数据,得到包括肿瘤细胞在内的9个细胞群的多条精确的lncRNA-miRNA-mRNA调控轴。生存分析结果显示,ITGA2、PDLIM5H和PRRG4低表达组的无病生存期显著高于高表达组（P<0.05）,免疫组织化学验证了上述基因在肿瘤组织中均有不同程度的表达。本研究成功构建了PCa单细胞水平的lncRNA-miRNA-mRNA调控网络,并发现其不仅参与PCa的发生和发展,同时在临床预后预测方面也有重要意义。     

MHC polymorphism and disease-resistance to Edwardsiella tarda in six turbot (Scophthalmus maximus) families

This study examined genetic variation in the major histocompatibility complex(MHC) Class II B gene in turbot(Scophthalmus maximus) by virulent bacterial pathogen challenge.One hundred fry from each of six families were infected with Edwardsiella tarda by intraperitoneal injection.Family mortality ranged from 28.0% to 83.3%.Complete exon 2 and intron 1 sequences of MHC Class II B genes were amplified from five survivor and five non-survivor individuals per family using the clone-sequence method.Thirty-seven sequences from 60 individuals revealed 37 different alleles,25 of which were unique to this study.The 25 unique alleles belonged to 16 major allele types.Nine alleles were used to examine the association between alleles and resistance/susceptibility to disease.Five alleles were present in an individual,suggesting a minimum of three loci or copies of the turbot MHC Class II B gene.The rate of non-synonymous substitution(d N) was 2.30 and 1.58 times higher than synonymous substitution(d S) in the peptide-binding regions(PBR) and non-PBR in whole families,respectively,which suggested balancing selection on exon 2 of the MHC Class II B gene in turbot.One allele,Scma-DBB1*02,was significantly more prevalent in survivor stock than in non-survivor stock(P=0.001).Therefore,this allele might be associated with resistance to bacteria.A second allele,Scma-DBB1*10,was significantly more prevalent in non-survivor stock(P=0.021),and is likely associated with susceptibility to bacteria.     

RACK1依赖的miRNA途径参与调节植物叶绿素的生物合成

为深入探索活化C激酶受体1(receptor of activated C kinase 1,RACK 1)在调节植物microRNA(miRNA)的生物发生,以及其靶基因中的关键作用,对在美国国家生物技术信息中心(National Center for Biotechnology Information,NCBI)网站上共享的基因表达综合数据库(Gene Expression Omnibus,GEO)中有关rack1突变体的小RNA序列数据重新进行了系统分析和挖掘,找到了19个新miRNA.通过解析差异miRNA表达,鉴定到了特异调控叶绿素合成相关基因HEMA和HEMC表达的miRNA,为今后深入系统地研究RACK1在调节叶绿体发育和光合作用机理提供了关键的理论依据,也为利用公共数据库挖掘潜在的数据信息提供了基本的研究设想和思路.     

AGO1基因对拟南芥叶边缘锯齿状发育的影响

在拟南芥和水稻中Argonaute(AGO)蛋白是RNA介导的沉默复合体(RISC)的核心组分,在植物叶极性的分化方面具有重要的调节作用。实验根据AGO1基因序列设计一对特异引物,提取野生型拟南芥RNA作为模板,采用反转录PCR方法扩增出AGO1基因,并插入到克隆载体pGEM-T中。筛选鉴定后将AGO1连接到植物表达载体pBI121上,构建起植物表达载体pBI121-AGO1。并且利用农杆菌介导的方法转化拟南芥,通过抗性筛选,获得转基因植株。然后对转基因植株进行表型分析及AGO1蛋白的RT-PCR检测。通过对转基因拟南芥表达分析发现,与野生型拟南芥相比超表达AGO1蛋白的转基因拟南芥叶片明显呈锯齿状,说明AGO1基因影响拟南芥叶片发育。     

Plant polar growth in tobacco disturbed by γ-tubulin gene silencing

To further understand the functions of γ-tubulin in plant cells, we conducted a study in which the γ-tubulin gene was down-regulated in tobacco plants (obtained by the Agrobacterium-mediated method). This involved transforming the target fragments, in which the sense and antisense partial γ-tubulin cDNA fragments were ligated together, into Nicotiana tabacum var. Samsun NN. The γ-tubulin downregulated transformants developed multiple meristems or branches with trumpet-shaped leaves; their root generation also appeared abnormal, with the taproots undeveloped, whereas lateral roots were developed. In addition, the content of indole-3-acetic acid (IAA) and expression of polarity transportation vector PGP1 were aberrant. These results suggest that γ-tubulin gene silencing disturbed the polar growth of tobacco plants, and that this phenomenon was probably correlated with the IAA content and the polar transportation process.     

The RNA splicing factor hSlu7 is required for correct 3' splice-site choice

The production of correctly spliced messenger RNA requires two catalytic splicing steps. During step II, exon 1 attacks an adenine-guanine (AG) dinucleotide at the 3' splice site. This AG is usually located between 18 and 40 nucleotides downstream from the branch site, and closer AGs are skipped in favour of AGs located more optimally downstream. At present, little is understood about how the correct AG is distinguished from other AGs. Here we describe a metazoan splicing factor (hSlu7) that is required for selection of the correct AG. In the absence of hSlu7, use of the correct AG is suppressed and incorrect AGs are activated. We investigated this loss of fidelity by analysing spliceosomes assembled in the absence of hSlu7. These studies reveal that exon 1 is loosely associated with these spliceosomes. Thus, the improperly held exon cannot access the correct AG, but can attack other AGs indiscriminately. We conclude that hSlu7 is required to hold exon 1 tightly within the spliceosome for attack on a prespecified AG.     

Crystal structure of opsin in its G-protein-interacting conformation

Opsin, the ligand-free form of the G-protein-coupled receptor rhodopsin, at low pH adopts a conformationally distinct, active G-protein-binding state known as Ops*. A synthetic peptide derived from the main binding site of the heterotrimeric G protein-the carboxy terminus of the alpha-subunit (GalphaCT)-stabilizes Ops*. Here we present the 3.2 A crystal structure of the bovine Ops*-GalphaCT peptide complex. GalphaCT binds to a site in opsin that is opened by an outward tilt of transmembrane helix (TM) 6, a pairing of TM5 and TM6, and a restructured TM7-helix 8 kink. Contacts along the inner surface of TM5 and TM6 induce an alpha-helical conformation in GalphaCT with a C-terminal reverse turn. Main-chain carbonyl groups in the reverse turn constitute the centre of a hydrogen-bonded network, which links the two receptor regions containing the conserved E(D)RY and NPxxY(x)(5,6)F motifs. On the basis of the Ops*-GalphaCT structure and known conformational changes in Galpha, we discuss signal transfer from the receptor to the G protein nucleotide-binding site.     

MiR-205 inhibits the invasion and migration of esophageal squamous cell carcinoma by modulating SMAD1 expression

Esophageal squamous cell carcinoma （ESCC） is one of the most lethal cancers worldwide. In this study, we aimed to investigate the underlying mechanisms of metastasis inhibition by miR-205 in ESCC. In microRNA （miRNA） array and quantitative RT-PCR analyses, we found that the expression level of miR-205 was significantly lower in patients with lymph node metastasis compared with that in patients without lymph node metastasis. After transfection of miR-205 mimics or inhibitors into ESCC cell lines, a significant negative correlation was observed between the expression level of miR-205 and Smad 1. In luciferase reporter assays, we revealed that miR- 205 inhibited the expression of SMAD1 by targeting the 3＇ untranslated region （3＇-UTR） of SMAD1 mRNA in ESCC cells. Furthermore, our results showed that miR-205 sup- pressed the invasion and migration of ESCC cells, whereas Smadl increased their invasion and migration. Taken together, our study demonstrates that miR-205 functions as a suppressor of tumor metastasis by regulating SMAD1 expression through targeting the 3＇-UTR of SMAD1 mRNAin ESCC. Therefore, miR-205 may be a potential therapeutic target for miRNA-based therapy of ESCC.     

Salicylic acid and ethylene signaling pathways are involved in production of rice trypsin proteinase inhibitors induced by the leaf folder Cnaphalocrocis medinalis （Guenee）

The roles of signaling pathways in the production of trypsin proteinase inhibitors (TrypPIs) in rice infested by the leaf folder (LF) Cnaphalocrocis medinalis were studied. Infestation by LF increased TrypPI levels in the leaves of rice plants at the tillering, booting and flowering stages but decreased TrypPI levels at the ripening stage; TrypPI levels in rice stems did not increase at any developmental stage. Infestation by LF at the tillering stage systemically increased TrypPI levels in leaves but not in stems; it also enhanced salicylic acid (SA) levels in leaves and stems, and the ethylene level released from plants. However, LF infestation did not increase JA concentrations. Exogenous application of SA or ethylene enhanced TrypPI levels in the leaves and stems of plants at the tillering stage, whereas treatment with both SA and ethylene induced lower levels of TrypPIs than treatment with SA or ethylene alone, suggesting an antagonistic effect of SA and ethylene on TrypPIs induction. The results suggest that both SA and ethylene signaling pathways are involved in the production of TrypPIs in rice induced by LF; moreover, the antagonistic effect of SA and ethylene may explain the changes in TrypPI levels seen at different plant developmental stages and in different organs.