糙皮侧耳原基期差异表达基因分析

为解析糙皮侧耳原基期与菌丝期差异表达的基因,本研究以原基期c DNA为检测子(tester)、双核菌丝期c DNA为驱赶子(driver),采用抑制性消减杂交法(suppression subtractive hybridization,SSH)构建了糙皮侧耳SSH c DNA文库。菌液PCR验证SSH c DNA文库插入c DNA片段后,挑取了2 055个差异转化子,差异转化子经3次反向Northern杂交筛选,得423个信号差异显著的克隆;阳性克隆测序后,经NCBI数据库Blastn和Blastx比对,共得206条差异表达序列(expressed sequence tag,EST),重复序列去除后,有46个基因参与了细胞急救和防御、能量代谢、转录和蛋白调控、膜蛋白和信号转导,18个基因编码未知功能的推定蛋白,5个无任何同源性的新基因。挑取10个差异表达基因进行半定量RT-PCR,发现这些序列在原基期的表达水平显著高于菌丝期。结果表明,本研究成功构建了糙皮侧耳原基期与菌丝期SSH c DNA文库,为进一步分离糙皮侧耳生长发育相关基因并研究糙皮侧耳的发育机制奠定了基础。

hBDNF基因在成纤维细胞中的异位表达

将 h BDNF全长编码序列插入人 I型胶原基因 (Colia1)增强子 -启动子调节序列之下 ,构建了含 Colia1-BDNF微基因的重组体 p SCEPBFCAT,并转染人胚肌腱成纤维细胞。成功地利用了 Colia1基因调控元件介导 BD-NF基因在成纤维细胞中异位表达 ,SDS- PAGE显示表达产物分子量为 2 7k Da,免疫斑点杂交、Elisa和 Western杂交证实该表达产物具有

溃疡性结肠炎异位表达PS_2蛋白的检测及意义

目的 探讨 PS2 蛋白在溃疡性结肠炎组织中异位表达的意义。方法 用免疫组织化学法检测了30例溃疡性结肠炎和 2 8名正常人结肠组织中 PS2 和 CEA的表达。结果 显示 30例溃疡性结肠炎 PS2 蛋白阳性的 7例 ,阳性率 2 3.3% ,PS2 只在伴有不典型增生的溃疡性结肠炎中表达 ;而 CEA阳性的 2 2例 ,阳性率 73.3%。2 8名正常人结肠组织中 PS2 阳性的 1例 ,CEA阳性的 2例。结论 PS2

益气养阴活血配伍对缺血心肌差异基因表达的影响

本研究在既往建立的大鼠缺血心肌基因差异表达谱基础上,探讨了益气养阴、活血及其配伍中药对缺血心肌差异基因表达影响及可能的作用机制.采用冠状动脉左前降支结扎法制备大鼠AMI模型,造模后将大鼠随机分为5组:模型组、美插洛尔组、益气养阴组、活血组和益气养阴活血组,并设正常组和假手术组,假手术组只穿线不结扎,术后24h给予正常组和假手术组等体积生理盐水,给予其他各组相应药物灌胃.第8天截取心肌缺血区组织,光学显微镜观察病理形态,分别应用荧光定量PCR及化学比色法检测目标基因mRNA表达水平和翻译蛋白酶活性.结果显示,模型组HE染色可见心肌组织缺血区心肌肿胀、炎细胞浸润和细胞溶解,各用药组病理改变较模型组均明显较轻,益气养阴组、活血组能量代谢相关基因细胞色素C氧化酶5a(Cox5a)和ATP合酶5e(ATP5e)基因表达量和酶活性均出现了下调趋势,而益气养阴活血配伍组Cox5a,ATP5e对应的蛋白酶活性显著下降(P<0.05).这表明能量代谢作用通路相关基因的异常表达是引起AMI心肌损伤的重要分子机制之一,益气养阴活血配伍中药影响COX5a,ATP5e等能量相关功能基因的表达是其抗心肌缺血的可能机制,较单纯益气养阴、活血效果理想.

Integrative analysis of bone-formation associated genes and immune cell infiltration in osteoporosis, and the prediction of active ingredients in targeted traditional Chinese medicine

Objective To explore the differential expression and mechanisms of bone formation-related genes in osteoporosis(OP)leveraging bioinformatics and machine learning methodologies;and to predict the active ingredients of targeted traditional Chinese medicine(TCM)herbs.Methods The Gene Expression Omnibus(GEO)and GeneCards databases were employed to conduct a comprehensive screening of genes and disease-associated loci pertinent to the pathogenesis of OP.The R package was utilized as the analytical tool for the identification of differentially expressed genes.Least absolute shrinkage and selection operator(LASSO)logis-tic regression analysis and support vector machine-recursive feature elimination(SVM-RFE)algorithm were employed in defining the genetic signature specific to OP.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses for the selected pivotal genes were conducted.The cell-type identification by estimating rela-tive subsets of RNA transcripts(CIBERSORT)algorithm was leveraged to examine the infiltra-tion patterns of immune cells;with Spearman’s rank correlation analysis utilized to assess the relationship between the expression levels of the genes and the presence of immune cells.Coremine Medical Database was used to screen out potential TCM herbs for the treatment of OP.Comparative Toxicogenomics Database(CTD)was employed for forecasting the TCM ac-tive ingredients targeting the key genes.AutoDock Vina 1.2.2 and GROMACS 2020 softwares were employed to conclude analysis results;facilitating the exploration of binding affinity and conformational dynamics between the TCM active ingredients and their biological targets.Results Ten genes were identified by intersecting the results from the GEO and GeneCards databases.Through the application of LASSO regression and SVM-RFE algorithm;four piv-otal genes were selected:coat protein(CP);kallikrein 3(KLK3);polymeraseγ(POLG);and transient receptor potential vanilloid 4(TRPV4).GO and KEGG pathway enrichment analy-ses revealed that these trait genes were predominantly engaged in the regulation of defense response activation;maintenance of cellular metal ion balance;and the production of chemokine ligand 5.These genes were notably associated with signaling pathways such as ferroptosis;porphyrin metabolism;and base excision repair.Immune infiltration analysis showed that key genes were highly correlated with immune cells.Macrophage M0;M1;M2;and resting dendritic cell were significantly different between groups;and there were signifi-cant differences between different groups(P<0.05).The interaction counts of resveratrol;curcumin;and quercetin with KLK3 were 7;3;and 2;respectively.It shows that the interac-tions of resveratrol;curcumin;and quercetin with KLK3 were substantial.Molecular docking and molecular dynamics simulations further confirmed the robust binding affinity of these bioactive compounds to the target genes.Conclusion Pivotal genes including CP;KLK3;POLG;and TRPV4;exhibited commendable significant prognostic value;and played a crucial role in the diagnostic assessment of OP.Resveratrol;curcumin;and quercetin;natural compounds found in TCM;showed promise in their potential to effectively modulate the bone-forming gene KLK3.This study provides a sci-entific basis for the interpretation of the pathogenesis of OP and the development of clinical drugs.

Transcriptome Sequencing for Sugar and Flavonoid Metabolism in Prunus persica‘Jinxiangyu’

In this study,high performance liquid chromatography(HPLC)and RNA-seq transcriptome sequencing were used to study the changes in soluble sugar components and flavonoids in Prunus persica‘Jinxiangyu’at different developmental stages(20–90 d after flowering)and screen the key genes regulating the formation of soluble sugar and flavonoids in the fruits.The results showed that 60–85 d after flowering was the key stage of quality formation of Prunus persica‘Jinxiangyu’,and the content of soluble sugar,soluble solid,fructose,and sucrose in the fruit increased significantly during this period.The sugar content of ripe fruits was mainly fructose and sucrose.The content of kaempferol glycoside was low in the fruit.Quercetin glycoside content was higher in the young fruit stage and decreased with fruit maturity.There were no anthocyanin compounds in the fruit.The expression levels of genes involved in flavonoid metabolism(ANS,DFR,F3H,FLS,4CL1,etc.)were low in the fruit.A total of 181 differentially expressed genes were identified during fruit development to participate in five sugar metabolism pathways,among which the SDH gene had a higher expression level,which continuously rised in the later stage of fruit development.It mainly promoted the accumulation of fructose content in the later stage of fruit development.The expression levels of SPS1,SS,and SS1 genes were continuously up-regulated,which played a key role in sucrose regulation.The higher expression levels of SUS3 and INVA genes in the early stage of fruit development promoted the degradation of sucrose.

Alterations of Root and Fiber in Transgenic Cotton Plants with Chimeric Ph/P-ipt Gene Expression

The seed_specific phaseolin promoter (Ph/P) was fused to an ipt gene, then was cloned to a plant expression vector containing a gus gene driven by a 35S promoter. Cotton (Gossypium hirsutum L.) plants were transformed through pollen tube pathway methods. After seed germination, histochemical staining of the roots demonstrated that 32 GUS positive plants were obtained and three of which contained the chimeric Ph/P_ ipt transgene as confirmed by PCR analysis. An immunosorbent assay showed that two of the three transgenic cotton lines contained higher levels of zeatin equivalents in seeds than the control. Seedling development of these two transgenic lines differed from the control in a reduction of the shoot growth, showing a stunted phenotype as expected, but a surprisingly developed root system with a 3-4 fold fast_growing lateral roots. In addition, fibers (seed_hairs) of the two transgenic cotton lines were considerably shorter than those of the control. These results indicate that genetic engineering may be used to manipulate the development of cotton plants, particularly cotton fibers.

Cloning and Differential Expression of a 1-Aminocyclopropane-1-Carboxylate Synthase cDNA from Peach

The ACC synthase is the key enzyme in ethylene biosynthesis and fruit ripening. To study the mechanism of ACC synthase in peach Prunus persica (L.) Batsch) fruit ripening, we cloned a full_length cDNA of ACC synthase pacs from peach using 5′/3′ RACE PCR. The nucleic acid sequence of pacs was 1 848 bp, containing 177 bp of 5′untranslated sequence, 1 449 bp of an open reading frame, and 219 bp of 3′untranslated sequence (excluding the stop codon TAA). The pacs open reading frame encoded a 483_amino acid polypeptide with a predicted size of 54 kD and a calculated PI of 6.43. The deduced protein from ACC synthase cDNA pacs had 65%, 70%, 75%, and 90% homology with the other deduced proteins from tomato (S19677), plum (AB031026), papaya (U68216) and apple (AB034993), which contained the active site of ACC synthase SLSKDMGFPGFR conserved among these plant ACC synthases. RNA_based PCR amplification combined with hybridization analysis with pacs and another ACC synthase cDNApacs12 (AF467782) cloned by us before as probes, indicated that expression patterns of both clones were very similar. mRNAs of both clones expressed in the alabastrum and petal, and were induced after ethylene treatment. Wounding and IAA treatments could induce ACC synthase expression of both clones in the leaves. However, the wounding treatment of leaves has induced more abundant pacs ACC synthase expression than that ofpacs12. Pacs mRNA expressed in both green mature and ripening fruit, whilepacs12mRNA was little or undetectable in green mature fruit, but apparent in ripening fruit. Both clone mRNAs accumulated more in leaves (following wounding and IAA treatments) and flowers than in fruits.

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.

Cloning of cDNA Encoding COMT from Chinese White Poplar ( Populus tomentosa ), Sequence Analysis and Specific Expression

The cDNA fragment encoding caffeic acid 3_O_methyltransferase (COMT) in Chinese white poplar ( Populus tomentosa Carr.) was isolated and cloned by RT_PCR technique. The size of the cDNA fragment is 1 080 bp, which almost covers the whole cDNA_encoding region. Authors’ cDNA fragment in P. tomentosa shares 98.7% homology with the reported corresponding cDNA in the P. tremuloids at nucleotide level, 99.4% homology at amino acid level, respectively. The analysis of Northern dot hybridization showed that COMT is expressed specifically in the developing secondary xylem of stem during the season of xylem differentiation, which means the linkage between the gene expression for a monolignol biosynthetic enzyme and seasonal regulation of xylem development in woody plant.

Analysis of Seed-specificity of Silencing fad_2 Gene Expression in Transgenic Rapeseed Line W-4(Brassica napus L.)

This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.

Gene Chip Analysis for Rice MicroRNA Response to Low Energy N^+ Beam Radiation

[Objective] The aim of this study was to explore the expression differences of miRNA response to low energy N＋ beam radiation in rice.[Method]Three groups of ion beam irradiation rice seeds and untreated rice seeds were selected respectively,and then the total RNA of seedlings after 96 h of germination was extracted at 30 ℃.Agilent gene chip was used to screen the differentially expressed genes of two groups of rice seedlings.The chip contained 510 miRNA probes;and about two times of expression differences between two samples were considered as the threshold range.[Result]14 miRNA molecules showed significant expression differences between the irradiated group and the control group,and all of them were decreased.[Conclusion]The miRNAs showed expression differences between irradiated group and the control group,which might regulate the expression of certain genes to respond to ion irradiation,thus producing physiological,biochemical and phenotypic differences.

Study on HSP70 Gene Expression in Different Tissue of Cyprinus carpio

[ Objective] The aim of this study was to investigate whether HSP70 can be used as a stress monitoring indicator in Cypnnus carpio breeding. [Method] Based on HSP70 sequence of Cyprinus carpio （AY120894）, one pair of primers was designed and synthesized, while the total RNA of liver tissues in Cyprinus carpio was extracted. Some cDNA fragments of Cyprinus carpio HSP70 were cloned by RT-PCR, and its differential expression in various tissues such as heart, intestine, mucus, gonad, swim bladder, gill and fin in Cyprinus carpio was also studied. [Result] The cDNA sequence of 480 bp was obtained from Cypdnus carpio HSPTO gene by RT-PCR amplification. Homology comparison between the deduced amino acid sequence after sequencing and that of other types of fish showed that the homology among Cyprinus carpio, Danio rerio, Ohcorhynehus mylciss, Paralichthys olivaceus, Xiphophoorus maculates and Carassius auratus was 96%, 98%, 98%, 96%, 98% and 96% respectively. The expression of HSP70 was detected in eight tissues of Cypnnus carpio. The expression was the highest in heart, followed by swim bladder and fin, but there was no significant difference between them （ P 〉 0.05 ）. There was no significant difference among the ex- pression in three tissues of intestine, mucus and fat （ P〉0.05）, but their expression was significantly higher than those in gonad and gill （ P〈 0.05）. [ Conclusion] HSPTO gene expression is a suitable criterion for monitoring the stress degree, stress capacity and healthy conditions in Cyprinus carpio breeding.

Genetic Transformation in Triticeae Crops

Wheat, triticale, tritordeum, barley, oat and rye are the most important crops in human consumptions and industry in the world. Transformation technology supplies a new source of improving Triticeae crops. In the past decade, transformation of wheat crops has considerably progressed. Many transgenic plants of Triticeae crops with various genes were produced via nricroprojectile bombardment, Agrobacterium-mediated transformation, PEG-uptake DNA technique, electroporation, microinjection, injection inflorescence and silicone carbide. Integration and expression of transgenes, inheritance and variation of transgenic plants have been studied. Technical improvements of genetic transformation for wheat crops will be extensively useful in commerce and benefit significantly to human being in the world.

Molecular Cloning and Characterization of the Tapetum- specific Gene RA39 from Rice

A novel tapetum-specific cDNA clone of rice, its corresponding gene designed as RA39, is isolated by RNA subtractive hybridization, differential screening and rapid amplification of cDNA ends. The RA39 cDNA is 1013 bp in length with an open reading frame encoding 298 amino acid residues. mRNA in situ hybridization reveals that RA39 is a tapetum-specific gene, and highly expressed at the meiosis stage of pollen mother cells. The deduced protein contains a signal peptide, a transmembrane region and a cytoplasmic tail, and is predicted to localize in endoplasmic reticulum by PSORT program. This cDNA sequence did not show significant homology to any known sequences in Genbank database. RA39 is the first gene identified to be expressed specifically in tapetal cells at the meiosis stage of pollen mother cells from cereals.

Differential Expression of Immune Genes between Body Side Skin and Groin Skin of Aohan Fine Wool Sheep

[Objective] To get major genes for wool traits regulation from immune genes. [Methods] Microarray technology was used to detect differentially expressed immune genes between body side skin （more wool growing） and groin skin （no wool growing） of Aohan fine wool sheep. [Results] 46 immune genes （fold change 〉2.0） were identified and classified, and then 6 of which were selected for QPCR confir- mation. The degree of consistency of the QPCR and microarray results was 66.67%, [Conclusion] Immune privilege may participate in wool growth regulation.

Site discrepancy of synonymous codon usage in SARS coronavirus and other viruses in Coronaviridae

The synonymous codon usage in the translational initiation and termination regions of genes of severe acute respiratory syndrome (SARS) coronavirus and five other viruses in Coronaviridae was systematically analyzed.The results indicate that most minor codons for these coronaviruses are preferentially used in the initial and terminal region.The minor codons preferentially used in the initial region are thought to have a negative effect on gene expression,which can be explained by the minor codon modulator hypothesis.It also indicates that the minor codons preferentially used in the terminal region may regulate the level of gene expression.The proposed results strongly imply that the minor codon modulator hypothesis can be applied to both some bacteria and some viruses.

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.

Molecular Characterization of Four ADF Genes Differentially Expressed in Cotton

Actin depolymerizing factor （ADF）, highly conserved in all eukaryotic cells, is a low molecular mass of actin-binding protein, which plays a key role in modulating the polymerizing and depolymerizing of the actin filaments. Four cDNAs （designated GhADF2, GhADF3, GhADF4, and GhADF5, respectively） encoding ADF proteins were isolated from cotton （Gossypium hirsutum） fiber cDNA library. GhADF2 cDNA is 705 bp in length and deduces a protein with 139 amino acids. GhADF3 cDNA is 819 bp in length and encodes a protein of 139 amino acids. GhADF4 cDNA is 804 bp in length and deduces a protein with 143 amino acids. GhADF5 cDNA is 644 bp in length and encodes a protein of 141 amino acids. The molecular evolutionary relationship of these genes was analyzed by means of bioinformatics. GhADF2 is closely related to GhADF3 （99% identity） and PetADF2 （89% identity）. GhADF4 is closely related to AtADF6 （78% identity）, and GhADF5 is closely related to AtADF5 （83% identity）. These results demonstrated that the plant ADF genes are highly conserved in structure. RT-PCR analysis showed that GhADF2 is predominantly expressed in fiber, whereas, GhADF5 is mainly expressed in cotyledons. On the other hand, it seems that GhADF3 and GhADF4 have no tissue specificity. Expression levels of different ADF genes may vary considerably in the same cell type, suggesting that they might be involved in regulating tissue development of cotton and the each ADF isoform may diverge to form the functional difference from the other ADFs during evolution.