mRNA differential display on gene expression in settlement metamorphosis process of Ruditapes philippinarum larvae

The mRNA differential display (DDRT-PCR) technique was adopted to find out the genes related tosettlement metamorphosis development process of Ruditapes philippinarum larvae.In this study,we haveobtained three hundred and forty-six amplification bands in total from pediveliger larvae,veliger larvae,eye spot larvae and post-larvae.Sixty-five out of three hundred and forty-six bands are distinctly differen-tial display from band pattern,which can be put into four groups,standing for different expression char-acters.Sixteen differential display bands were cloned,sequenced and analyzed and nine different se-quences are obtained in the study.Three sequences have higher similarity to the cDNAs deposited indatabase and three are very similar to the rDNA of other species,considered as the rDNA of Ruditapesphilippinarum.The rest three sequences are found to be novel sequences after analyzed.Their accessionnumbers are AY916799,AY916798,and AY916797 respectively.We thought the novel sequences arepossibly relevant to the early embryo development of Ruditapes philippinarum larvae and can provide somefundamental understandings that are helpful for the improvement of scallop seed raising industry.

Differential Gene Expression Between Wheat Hybrids and Their Parental Inbreds in Primary Roots

To provide an insight into the molecular basis of heterosis, differential display of mRNA was used to analyze the difference of gene expression between wheat (Triticum aestivum L.) heterotic hybrid A, nonheterotic hybrid B and their parental inbreds in the primary roots. By using 5′ end random primers in combination with three one-base-anchored primers, it was found that 22.5% and 22.9% of 877 total displayed cDNAs were differentially expressed between hybrid A, B and their parents, respectively. Both quantitative and qualitative differences in gene expression between hybrids and their parental inbreds were obvious, indicating that the patterns of gene expression in hybrids alter significantly as compared to their corresponding parents. On the other hand, by using MADS-box gene specific 5′ end primer for DDRT-PCR, we found that nearly all of the displayed cDNA fragments were polymorphic between hybrids and their parents, and major difference occurred in qualitative level, in which hybrid specific-expressed and silenced genes are the major two patterns, suggesting that MADS-box gene may be important for manifestation of differential gene expression and wheat heterosis. In comparison with our previous results by using seedling leaves, it is indicated that differential gene expression between hybrids and parents is dependent on the tissues tested, and more differentially expressed genes were observed in the primary roots than in the seedling leaves. Therefore, it is concluded that the expressions of both randomly displayed cDNAs and transcription factor genes, such as MADS-box, alter significantly between hybrids and their parents, which might be responsible for the observed heterosis.

Relationship Between Differential Gene Expression and Heterosis During Ear Development in Maize (Zea mays L.)

Maize （Zea raays L.） is one of the most important crops because of the remarkable properties of its hybrid, which is responsible for the high commercial value of hybrid maize. The genetic basis of heterosis （hybrid vigor） is not well understood. A differential display technique was performed to identify genes with differential expression across twelve maize inbred lines and thirty-three hybrids during ear development. An incomplete diallel design was used to investigate the relationship between the global framework of differential gene expression and heterosis. It was found that the genes belonging to MONO pattern （i.e., genes expressed in both parental lines and in hybrid） was the highest in percentage among the total five patterns and illustrated that the properties of differentially expressed genes are not entirely responsible for heterosis. Furthermore,a larger number of differentially expressed genes in hybrid, which serves as a major reservoir for generating novel phenotypes that exhibit heterosis of certain agronomic traits during early development and differentiation of maize ear. Moreover, there were some silent genesin hybrids that are responsible for the arrest or abortion of spikelets and for the increase in kernels weight.

Differential Gene Expression Between Hybrids and Their Parents During the Four Crucial Stages of Cotton Growth and Development

The study aims to clarify the differential gene expression between cotton hybrids and their parents in order to better understand the molecular basis of cotton heterosis. The research focused on cotton heterotic and lower heterotic hybrids and their parents during the four crucial stages, which were analyzed using a differential display technique. The results indicated that there were both quantitative and qualitative differences in gene expression amongst them. The quantitative differences include over- and under-expression of parental genes and the dominant expression of highly-expressed parental genes in hybrids. In contrast, the qualitative differences are the following： （i） Bands were observed in both parents but not in the F1 hybrid （BPnF1）; （ii） bands occurred in either of the parents but not in the F1 hybrid （UPnF1）; （iii） bands presented only in the F1 hybrid but not in either of the parents （UF1nP）; and （iv） bands were detected in either of the parents and the F1 hybrid （UPF1）. Overall, the major differences of gene expression occurred in the qualitative level and four related differential patterns were observed. Furthermore, the amount of differential patterns during the flowering stage was relatively higher than those of other stages. At this juncture, both the amount of hybrid-specific expression patterns at flowering stage and the silenced expression patterns at boll-forming stage in highly heterotic hybrids were found higher than those in the lower heterotic ones. It was concluded that significant differences of gene expression in leaves were present between cotton hybrid and its parents during the whole growing stages. Hence, these differences might be responsible for the observed cotton heterosis.

Isolation, Identification and Tissue Expression Profile Analysis of One Novel Differentially Expressed Sequence Tag in the Longissimus dorsi Muscle from Meishan, Meishan × Large White Hybrid and Large White Pigs

In order to detect the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performed to investigate the differences of gene expression in the Longissimus dorsi tissue from Meishan, Meishan × Large White hybrid and Large White pigs with nine 3'-end anchored primers in combination with ten 5'-end arbitrary primers and nearly 3000 reproducible bands were examined. One novel expressed sequence tag (EST4, GenBank accession number: AY553914) that was differentially expressed in Meishan, Meishan× Large White hybrid and Large White pigs was isolated from the Longissimus dorsi muscle tissue and identified through semi-quantitative RT-PCR. BLAST analysis revealed that the 350 bp long EST (EST4) was not homologous to any of the known porcine genes. Tissue expression profile analyses showed that the EST4 was expressed in most of tissues.LIU Yong-gang, Ph D candidate

Gene expression profiles associated with osteoblasts differentiated from bone marrow stromal cells

Objective:To study the changes of gene expression profiles associated with osteoblasts differentiated from rat bone marrow stromal cells in vitro by gene chip technique.Methods:rat Rone marrow stromal cells were isolated and cultured,and differentiation was induced by dexamethasone,β-glycerol phosphate and vitamin C.Cellular mRNA was extracted and reverse transcribed into cDNA,thus related genes expression differences were detected by gene expression profile chip.Results:Calcifying nodules were visible in the induced cells.There were27.7%genes expressed differentially,three times more than the normal and induced cells,and some genes were related to transcription,translation,glycosylation modification.Extracellular matrix,signal molecules and metabolism were up—regulated.Conclusions:The gene chip technique can be used to detect the multi-gene different expression in the differentiationinduceed rat BMSCs,and these differentially expressed genes are necessary genes related to rat BMSCs proliferation and induction of osteoblastic differentiation.

Expression profiles of genes regulated by BplMYB46 in Betula platyphylla

The transcription factor BplMYB46 has been identified as a regulator of abiotic stress responses and promoter of secondary wall deposition in Betula platyphylla.To investigate the downstream targets of BplMYB46,the expression profiles of genes in stems from BplMYB46-overexpressing(OE)and BplMYB46-silencing(SE)plants were studied.In OE stems,952 genes were upregulated,and 1469 were downregulated in comparison to SE stems.In a KEGG pathway enrichment analysis of differentially expressed genes(DEGs),1387 differentially expressed genes were annotated for 117 metabolic pathways.DEGs were abundant for metabolic pathway,secondary metabolite biosynthesis,plant hormone signal transduction,phenylpropanoid and flavonoid biosynthesis.DEGs were implicated lignin or cellulose biosynthesis,cell wall modification,xylem development,disease resistance,stress responses,and anthocyanin biosynthesis.These results suggested that BplMYB46 regulates cell wall development and stress resistance by affecting the expression of these genes.Our study further elucidates the mechanism by which BplMYB46 mediates abiotic stress responses and secondary cell wall biosynthesis in birch.

An Integrated Technique for Identification of Differential Genes Expressed in Patients with Cancer

Summary: To develop a method for identification of differential gene expression between different cell populations, several convenient techniques of molecular biology, including subtractive hybridization, suppression PCR, T/A cloning and sequencing, were used to identify genes expressed differentially in CD45+ and CD45-cells isolated from U266 cell line of multiple myeloma. Our results showed that the levels of abundant genes scale down 20 times through subtractive hybridization. Plasmid DNA from CD45+ cell clones was hybridized with forward or backward cDNA probes synthesized from CD45+ and CD45-cells, respectively. A few of differentially expressed genes reconfirmed by RT-PCR were identified from 500 expressed clones of CD45+ cells. It is concluded that a strategy for gene expression identification developed from conventional molecular biological methods can be used in different laboratories.

Oligomicroarray-based primary study of gene expression profile changes in Barrett’s esophagus

Objective: To analyze the differential expression genes (DEGs) between Barrett’s esophagus (BE) and normal esophagus mucosa and explore the target genes related to the development and progression of BE. Methods: The total RNAs of matched BE and normal esophagus mucosa of BE patients were isolated using one step Trizol method. Matched RNAs were qualified using 10 g/L agarose gel electrophoresis. cRNAs were synthesized, fluorescence labeled and purified after total RNAs were purified. The RNAs of BE and normal esophagus mucosa were hybridized with Agilent oligomicroarray (30 968 probes). The fluorescence intensity features were detected by Agilent scanner and quantified by feature extraction software. Results: (1) The total RNA, reverse transcription product and fluorescence labeled cRNA were all of high quality; (2) There were 142 up-regulated genes and 284 down-regulated genes among 2-fold DEGs. Conclusion: Microarray-based studies are feasible in endoscopically obtained tissues. Many BE-associated genes are screened by the high-throughput gene chip. The development and progression of BE is a complicated process involving multiple genes and multiple procedures, and functional study of these genes may help to identify the key genes or pathways involved in the pathogenesis and development of BE.

Screening the stage-specific expression gene from different germ cells of rat

Objective: To screen the stage-specific expression genes from rat spermatogonia, pachytene spermatocytes and round spermatids. Methods: Highly purified spermatogonia were isolated from 9-day-old rats, pachytene spermatocytes and round spermatids from adult rats by sedimentation velocity at unit gravity, using 2%-4% BSA gradient in DMEM/F12 medium. A mRNA differential display method was used for screening the stage-specific expression gene. Results: Nineteen differentially expressed cDNA fragments were obtained. After excluding the false positive cDNA fragments by dot blot, 13 cDNAs were selected to clone and sequence. To obtain longer cDNAs, six ESTs were used to screen the rat testis λ-zap II cDNA library. Two longer cDNA fragments, designated as LY21 and LM66, were obtained. The analysis with DNAMAN software indicated that LY21 had a long open reading frame coding 372 amino acids while LM66 had no long open reading frame. LY21 were highly homologous with hnRNP H1. To observe the expression patterns of LY21 gene in the testicular cells, we performed in situ hybridization on testis sections from adult rats. The LY21 gene expression was found in the spermatogonia and primary spermatocytes.Conclusion: This study indicated that LY21 gene was associated with spermatogenesis. Further studies will be needed to explore the function of LY21.

Active Methyl Cycle and Transfer Related Gene Expression in Response to Drought Stress in Rice Leaves

Three rice varieties, Zhonghan 3, Shanyou 63 and Aizizhan, were used as materials in detecting differential active methyl cycle and transfer related gene expression in response to drought stress. The experiment was performed by gene chip and mRNA differential display technologies under the conditions of drought simulated with 10% PEG6000 solution. The results indicated that the methyl cycle could be activated in the leaves of Zhonghan 3 and Shanyou 63 but inhibited in the leaves of Aizizhan under drought stress. Furthermore, drought stress could induce the expression of a large number of methyltransferase genes, especially the transcription of Rubisco protein methylation related genes, which are beneficial for prevention of Rubisco protein oxidation and degradation, and drought stress could inhibit the transcription of DNA methyltransferase genes and histone methyltransferase genes. This result confirmed that the active methyl cycle and transfer related genes were involved in rice drought resistance.

Differential Gene Expression Profiles in Coronary Heart Disease Patients of Blood Stasis Syndrome in Traditional Chinese Medicine and Clinical Role of Target Gene

Objective:To investigate the differential gene expression profiles in coronary heart disease(CHD) patients of blood-stasis syndrome(BSS) by oligonucleotide microarray technique,and the clinical significance of target gene.Methods:Subjects were assigned to CHD patients with BSS(n=8),CHD patients without BSS (n=8),and BSS patients without CHD(n=8) based on coronary angiography and the diagnostic criteria of BSS. The sex- and age-matched healthy volunteers(n=8) were enrolled as the control group.Venous blood s...

Primarily screening and analyzing ESTs differentially expressed in rats' primary liver cancer

Objective： To screen and analyze key express sequence tags （ESTs） which were differentially displayed in every period of SD rats＇ primary hepatic carcinoma and reveal the molecular mechanism of carcinogenesis. Methods： Using diethylnitrosamine （DENA） as a cancerigenic agent, animal models with different phases of primary hepatic cancer were constructed in SD rats. Rats were respectively sacrificed at d 14, d 28, d 56, d 77, d 105 and d 112 after the rats received DENA by gavage, then the livers were harvested. One part of the livers was classified according to their pathological changes, while the other was reserved for molecular mechanism studies on hepatocarcinogenesis. The differentially expressed genes were isolated from both normal and morbid tissues by mRNA differential display technique （DDRT-PCR）. After the fragments were sequenced, bioinformatics were .used to analyze the results. Results： Twelve differentially expressed cDNA fragments were obtained. Nine fragments had the homology with known cDNA clones, especially EST-7 was similar to BN/SsNHsdMCW mitochondrion gene and the identity was 100% which suggested EST-7 may be the part of BN/SsNHsdMCW mitochondrion gene. In contrast, other three fragments （EST-1, EST-3 and EST-5） had extremely low identity to any genes registered in GENBANK databases. Conclusions： BN/SsNHsdMCW mitochondrion gene was expressed in different periods of hepatocarcinogenesis. Moreover, EST-I, EST-3 and EST-5 were suggested to contribute to the development of rat hepatocarcinogenesis, and thus may be candidates of new targets of oncogenes or cancer suppressor genes.

Characterization of expressed genes in the establishment of arbuscular mycorrhiza between Amorpha fruticosa and Glomus mosseae

Arbuscular mycorrhiza （AM） formed between plant roots and fungi is one of the most widespread symbiotic associations in nature. To understand the molecular mechanisms of AM formation, we profiled 30 symbiosis-related genes expressed in Amorpha fruticosa roots colonized by Glomus mosseae and in non-mycorrhizal roots at different stages using differential-display RT-PCR （DDRT-PCR）. The expressed genes were confirmed by reverse Northern blotting. Eleven fragments were sequenced and putatively identified by homologous alignment. Of the eleven AM-related genes, five were obtained at the early-stage of plant-fungus interaction and six at the later stage. Three expressed se-quence tag （ESTs） sequences were found to originate from the fungi and eight from the host plant by use of PCR evaluation of gDNA of both plant and fungi. The target genes included an ATP-binding cassette sub-family transporter gene, a transposon-insertion display band, and a photosynthesis-related gene. The results provided information on the molecular mechanisms underlying the development of mycorrhizal sym-biosis between woody plants and AM fungi.

A Rice CaMBP Gene is Induced in Organ-Specific Manner by Both Chilling and Heat-Shock Treatments

A rice CaMBP gene, OsCaMBP （AB363406）, was isolated from a chilling treated rice using the fluorescent differential display （FDD） screening method. Its cDNA sequence （2094 bp） contains an opening reading frame （ORF） encoding a 569 amino acids protein （63.2 kD）. OsCaMBP has the typical structural features of the CaMBP family, including the conserved IQ calmodulin-binding motif at the N-terminus. Homology analysis revealed 38.25%-47.28% identities of OsCaMBP with other CaMBPs in plants. RT-PCR analysis showed that the expression of OsCaMBP was remarkably inducible under the chilling （8℃） and heat-shock （42℃） treatments. OsCaMBP was undetectable under the normal conditions, and induced under the chilling treatment for 1 h, as well as the heat-shock treatment for 15 min, suggesting that the gene plays important roles in the signaling pathway in rice under both chilling and heat-shock stresses.

Identification of Candidate Genes Related to Polyploidy and/or Apomixis in <i>Eragrostis curvula</i>

This work was aimed at identifying genes that show altered expression profiles in response to changes in ploidy and/or reproductive mode (from sexual to apomictic) in the African grass Eragrostis curvula. A differential display analysis was performed on leaf and flower transcriptomes from a series of genetically related euploid plants, including tetraploid apomictic, diploid sexual, and tetraploid sexual plants. More than 100 primer combinations were used to generate 11,864 total markers, yielding 1293 differential bands. Of these bands, 11.84% to 6.74% were related to ploidy and 0.71% to 2.17% to the reproductive mode, depending on the tissue. A small percentage of bands showed similar expressions between the tetraploid apomictic and the diploid sexual plants. Expression-based similarity dendrograms were constructed. Our data suggested that ploidy is more decisive than tissue type in defining the transcriptome structure. Out of 102 fragments sequenced, 50 showed strong homology to known genes. The differentially expressed genes were mapped in silico onto maize chromosomes. Several candidates mapped within the linkage group syntenic to the Tripsacum dactyloides diplospory-governing region. The evidence indicates that expression of genes located around the diplospory-associated region may be strongly influenced by ploidy and may be silenced in the apomictic genotype. These findings are discussed in the context of diplospory molecular control and its connection with ploidy.

甘蓝型油菜BnMAPK1超量表达及中油821的转录差异表达分析

甘蓝型油菜(Brassica napus L.)是我国重要的油料作物之一,具有较高的产量及营养价值。挖掘油菜广谱抗逆基因并深入探究其分子机制,对提高油菜抗逆性且保证产量及品质具有重要意义。MAPK级联(Mitogen-activated protein kinase cascades)是多种信号跨膜传递的交汇点,参与生长发育、生物及非生物胁迫应答等生物学过程。为了解甘蓝型油菜BnMAPK1基因的生物学功能,本研究以BnMAPK1超量表达(OE)和中油821(CK)油菜为材料进行数字基因表达谱分析。结果显示,与CK相比,650个基因在OE油菜中差异表达;其中上调表达基因243个,下调表达基因407个。GO注释共富集到生物学过程118条途径,其中18条与光合作用相关(包含77个差异表达基因);分子功能7条途径,包括草酸氧化酶活性、锰离子结合、氧化还原酶活性等;细胞组分38条途径,包括叶绿体、质体、类囊体等。KEGG分析结果表明,BnMAPK1参与调控油菜光合作用、碳代谢、次生代谢物生物合成等14条过程。此外,利用实时荧光定量PCR验证8个候选基因在OE和CK油菜中的表达,其表达变化与测序结果基本一致。本研究结果为甘蓝型油菜BnMAPK1在生长发育及光响应等生物学过程中的功能分析奠定了基础,也为农作物广谱抗逆遗传育种提供了理论依据。

COVID-19患者外周血单个核细胞lncRNA相关ceRNA调控网络的生物信息学分析

目的:对2019冠状病毒病(coronavirus disease 2019,COVID-19)患者外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)测序数据进行生物信息学分析,分析长链非编码RNA(long non-coding RNA,lncRNA)的表达谱及竞争性内源RNA(competing endogenous RNA,ceRNA)调控网络,探讨其与COVID-19发病机制的关联。方法:利用R语言对从基因表达综合(Gene Expression Omnibus,GEO)数据库筛选的COVID-19相关测序数据进行基因差异表达分析并注释基因属性,鉴定出差异表达的lncRNA和信使RNA(messenger RNA,mRNA)。使用miRcode在线工具预测与差异表达的lncRNA相互作用的微RNA(microRNA,miRNA),再利用TargetScan、miRDB和miRTarBase数据库预测miRNA下游靶基因(mRNA),并与差异表达mRNA取交集,然后利用Cytoscape构建ceRNA调控网络。利用R语言对ceRNA调控网络中的mRNA进行基因本体论(Gene Ontology,GO)和京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析。结果:与健康人群相比,COVID-19患者PBMCs中313个lncRNA和1308个mRNA表达差异有统计学意义(P<0.05)。构建了ceRNA调控网络,在该网络中差异表达的lncRNA、mRNA分别有22、76个。富集分析发现:ceRNA网络内mRNA主要参与血管发育及生成的调控、(平滑)肌细胞增殖调控、上皮细胞凋亡过程、对缺氧的应答;黏着斑、细胞-基质黏附连接、紧密连接等;以及磷脂酰肌醇3激酶-蛋白激酶B(phosphatidylinositol 3 kinase-protein kinase B,PI3K-AKT)信号通路、丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路、流体剪切应力与动脉粥样硬化、糖尿病并发症中的晚期糖基化终产物-晚期糖基化终产物受体(advanced glycation end products-receptor for advanced glycation end products,AGE-RAGE)信号通路等。结论:本研究构建了lncRNA相关的ceRNA网络,为探讨lncRNA参与COVID-19发病机制提供了一个新的视角。这些基因有可能成为潜在的治疗靶点。

肿瘤坏死因子α预处理人脐带间充质干细胞的生物学特征分析

背景:在部分临床前及临床研究中,人脐带间充质干细胞表现出有希望的治疗结果但仍然存在效果有限的问题。因此,通过预处理的方式增强人脐带间充质干细胞的功能为干细胞培养工艺的开发提供新思路。目的:探索炎性因子肿瘤坏死因子α预处理人脐带间充质干细胞的生物学特性变化及作用机制。方法:分离获得的人脐带间充质干细胞培养至第6代,进行炎性因子肿瘤坏死因子α预处理,对比未预处理人脐带间充质干细胞和炎性因子预处理后人脐带间充质干细胞的免疫调节能力、抗炎功能、表面标志物、倍增时间、成脂成骨分化能力的变化,此外对比两组的转录组数据,分析具体机制。结果与结论:(1)与未预处理对照组相比,不同质量浓度肿瘤坏死因子α预处理的人脐带间充质干细胞均显著抑制T细胞增殖,促进Treg细胞增殖,其中1 ng/mL肿瘤坏死因子α作用效果最显著;(2)与未预处理对照组相比,不同质量浓度肿瘤坏死因子α预处理的人脐带间充质干细胞在体外炎症模型中释放更低水平的肿瘤坏死因子α和更高水平的白细胞介素10,其抗炎能力显著增强;(3)两组人脐带间充质干细胞的倍增时间、表面标志物表达无显著差异,均具有成骨成脂分化能力;(4)两组人脐带间充质干细胞的转录组变化主要体现在核因子κB信号通路和肿瘤坏死因子α信号通路的活化;(5)上述实验结果表明,肿瘤坏死因子α预处理后人脐带间充质干细胞的免疫调节及抗炎功能显著增强。

Effects of Panaxadiol Saponins Component as A New Chinese Patent Medicine on Proliferation,Differentiation and Corresponding Gene Expression Profile of Megakaryocytes

Objective： To investigate the effects of panaxadiol saponins component （PDS-C） isolated from total saponins of panax ginseng on proliferation, differentiation and corresponding gone expression profile of megakaryocytes. Methods： Bone marrow culture of colony forming assay of megakaryocytic progenitor cells （CFU-MK） was observed for the promoting proliferation mediated by PDS-C, and differentiation of megakaryocytic blasts caused by PDS-C was analyzed with flow cytometry in CHRF-288 and Meg-01 cells, as well as proliferation, differentiation-related genes expression profile and protein expression levels were detected by human gone expression microarray and western blot. Results： In response to PDS-C 10, 20 and 50 mg/L, CFU-MK from 10 human bone marrow samples was increased by 28.9± 2.7%, 41.0% ± 3.2% and 40.5% ± 2.6% over untreated control, respectively （P〈0.01, each）. Flow cytometry analysis showed that PDS-C treated CHRF-288 cells and Meg-01 cells significantly increased in CD42b, CD41, TSP and CD36 positive ratio, respectively. PDS-C induced 29 genes up-regulated more than two-fold commonly in both cells detected by human expression microarray representing 4000 known genes. The protein expression levels of ZNF91, c-Fos, BTF3a, GATA-1, RGS2, NDRG2 and RUNX1 were increased with western blot in correspond to microarray results. Conclusion： PDS-C as an effective component for hematopoiesis, play the role to enhance proliferation and differentiation of megakaryocytes, also up-regulated expression of proliferation, differentiation-related genes and proteins in vitro. KEYWORDS panaxadiol saponins, megakaryocyte, gone expression profile, proliferation, differentiation