高危人乳头瘤病毒58型E6基因的克隆及表达

目的 :获得含人乳头瘤病毒 (HPV) 5 8型E6基因的克隆及表达重组体并体外表达E6蛋白 .方法 :聚合酶链方法从 1例宫颈腺癌患者癌组织DNA中获得HPV5 8E6基因 ,并将其与克隆载体pGEM TEasy连接 ,获得重组体HPV5 8 E6 pGEM T ,继之以双酶切将E6基因与同样双酶切的线性化的pRSET A表达载体连接 ,得到E6表达重组体pRSET 5 8E6 ,转化E .coliBL2 1 (DE3) ,用IPTG诱导表达 .结果 :从 1例宫颈癌患者中成功获得了少见HPV5 8型的E6基因并构建了其重组表达载体 .经IPTG诱导后可表达Mr2 4 0 0 0的 6HisHPV5 8E6融合蛋白 ,表达量占菌体蛋白的 1 0 % .结论 :成功获得了少见HPV5 8高危型的E6基因 ,并可在E .

人乳头瘤病毒6型L1基因的克隆和表达及鉴定

克隆人乳头瘤病毒6型（HPV-6）保护性抗原L1基因。构建L1基因表达载体。从临床诊断尖锐湿疵的病理组织标本中提取HPV-6的DNA，采用高保真PCR扩增其保护性抗原L1基因，T-A克隆后测定核苷酸序列。构建pET32a的L1表达载体，在E．coli BL21DE3宿主菌中用不同浓度的IPTG诱导表达。采用SDS-PAGE鉴定表达产物。所克隆的L1基因与报道的相应核苷酸序列同源性为99．20％～99．93％，氨基酸序列同源性高达99．80％～100％。SDS-PAGE结果显示，在构建的载体中成功表达预计大小分子量的融合蛋白，成功构建了HPV-6的保护性基因L1的表达系统。

蜕膜细胞条件培养液对卵巢癌细胞株COCl侵袭相关基因表达的影响

目的 研究蜕膜细胞条件培养液(DCM)对卵巢癌相关侵袭基因表达的影响。方法 原代培养早孕蜕膜细胞及晚孕蜕膜细胞并提取DCM,用DCM处理卵巢癌细胞株COCl,利用RT-PCR法对卵巢癌侵袭相关基因的表达进行分析。结果卵巢癌细胞株COC1表达基质金属蛋白酶2(MMP-2)、基质金属蛋白酶抑制剂(TIMP-2)及纤溶酶原抑制剂(PAI-1),而不表达MMP-9、TIMP-1及尿激酶型纤溶酶原激活物(u-PA)。早孕及晚孕DCM能显著下调卵巢癌细胞株COC1 MMP-2的表达(P<0.01);早孕及晚孕DCM处理的卵巢癌细胞株COC1也表达TIMP-2、PAI-1 mRNA,且有显著差异(P<0.01)。结论DCM可以通过改变卵巢癌细胞株COCl侵袭基因MMP-2/TIMP-2以及B-PA/PAI-1之间的平衡,从而降低卵巢癌细胞株COCl的侵袭能力。

鼻咽癌患者外周血单个核细胞Fas和FasL的表达及其意义

目的:研究凋亡相关基因Fas和FasL在鼻咽癌患者诱导化疗前后外周血单个核细胞中表达水平的变化,探讨其在鼻咽癌发生、发展中的作用及意义。方法:采用流式细胞仪对38例鼻咽癌患者诱导化疗前后及15例正常健康对照组外周血单个核细胞中的Fas和FasL表达进行检测,并结合临床资料进行分析。结果: ( 1 )鼻咽癌患者外周血单个核细胞中Fas、FasL的表达明显高于正常健康对照组(P<0.05);Ⅲ+Ⅳ期患者的表达明显高于Ⅱ期患者(P<0.05)。(2)T3+4期患者外周血单个核细胞Fas表达明显高于T1+2期患者(P<0.05),而其FasL的表达稍高于T1+2期患者(P>0.05)。(3)N1 期患者的Fas表达明显高于N0 期患者(P<0.05),而其FasL的表达稍高于N0 期患者(P>0.05);N2+3期患者的Fas表达明显高于N1期(P<0 .05),而其FasL表达稍高于N0 期和N1 期(P>0.05)。(4)鼻咽癌患者经诱导化疗后,其Fas表达明显增高(P<0.05),FasL表达稍有增高(P>0.05)。结论:外周血单个核细胞Fas、FasL表达水平与鼻咽癌发生、增殖、局部转移有关,对其进行检测可作为鼻咽癌的诊断及病期判断的辅助指标。

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.

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.

Selection of Reference Genes in Transcription Analysis of Gene Expression of the Mandarin Fish, Siniperca chuasti

At present, transcription analysis of gene expression commonly uses housekeeping genes as control for normalization. In this study, the expression levels of three housekeeping genes including GAPDH, β-actin, and 18S rRNA in six tissues and five developmental stages of the Mandarin fish Siniperca chuatsi were assayed with quantitative real-time PCR （qPCR）. Differences in expression levels were analyzed using geNorm program. The results demonstrate that β-actin is the most stable gene at developmental stages and GAPDH is the most stable in different tissues. While 18S rRNA expression during development is differentially regulated, which indicates it is suitable as an internal control for gene expression normalization at the developmental level. Overall, the data suggest that the two most stable housekeeping genes are enough to accurately calibrate gene expression in S. chuatsi. The significance of this study provided convincing references and methodology for housekeeping gene selection and normalization in gene expression analysis with regular PCR or qPCR.

The Relationship of Methyl Jasmonate Enhanced Powdery Mildew Resistance in Wheat and the Expressions of 9 Disease Resistance Related Genes

[Objective] This study was carried out to determine the induction effect of jasmonic acid（JA）on powdery mildew resistance in wheat,the activation effect on the expressions of plant disease resistance related genes,and to investigate the relationship between the induced resistance and the gene expression patterns.[Method] Three powdery mildew susceptible cultivars of ＂Chinese Spring＂,＂Pumai 9＂ and ＂Zhoumai 18＂ typically representing different phenotypes in the field were employed.The powdery mildew was assessed by detached leaf assay,and real time quantitative RT-PCR was used to determine the expression patterns of 9 disease resistance related genes of PR1（PR1.1）,PR2（β,1-3 glucanase）,PR3（chitinase）,PR4（wheatwin1）,PR5（thaumatin-like protein）,PR9（TaPERO,peroxidase）,PR10,TaGLP2a（germin-like）and Ta-JA2（jasmonate-induced protein）in leaf of the three cultivars.[Result] MeJA application enhanced the powdery mildew resistances of ＂Chinese Spring＂,＂Pumai 9＂ and ＂Zhoumai 18＂.The induced powdery mildew resistance could be detected from 12 h to 96 h after MeJA treatment,and the peak value was at 24 h.Though there were differences between the three cultivars,MeJA significantly effect on the expressions of the 8 disease resistance related genes except TaGLP2a,and the peak values were at 12 h,24 h or 48 h after treatments.The strongest activation of MeJA was on PR9 and PR1 that their expressions could reach more than 100 times of the untreated samples.MeJA strongly activated PR2、PR4、PR5、PR3、PR10 and Ta-JA2,their expression could reach 10 to 70 times,and there was almost no activation effect on TaGLP2a.The induced powdery mildew resistance positively correlated with the induced expressions of the 8 disease related genes.[Conclusion] The induced powdery mildew resistance positively correlated with the induced expressions of the disease related genes.Jasmonate signalling plays a role in defence against Blumeria graminis f.sp.tritici.and future manipulation of this pathway may improve powdery mildew resistance in wheat.

Expression analysis of ThGLP, a new germin-like protein gene, in Tamarix hispida

Germin and Germin-like protein （GLP） have various proposed roles in plant developmental stages and stress- related processes. A novel GLP cDNA clone was isolated from a cDNA library of Tamarix hispida. ThGLP, coded 225aa, possesses conserved motif of plant germin and Germin-like protein. ThGLP belongs to true germin subfamily through phylogenetic analyses. Gene expression profiles in roots and leaves were evaluated using real-time quantitative RT-PCR. The results show that the gene was highly induced by drought, salt, low temperature, CdCl2 and abscisic acid treatments. Our results demonstrate that the ThGLP gene is expressed in leaves and roots, is involved in different abiotic stress re-sponses and controlled by an ABA-dependent signaling pathway.

Transformation of Pea Lectin Gene and Parasponia Haemoglobin Gene into Rice and Their Expressions

Lectin and leghemoglobin in legumes play the important roles, respectively, in recognition of host plants to their rhizobial bacteria, and lowering the oxygen partial pressure around bacteroids and protecting nitrogenase from oxygen in symbiotic nitrogen-fixing nodules. In order to extend the host range of the rhizobial bacteria and to make them fix nitrogen in non-legumes, pea lectin gene (pl) and Parasponia hemoglobin gene ( phl,) have been constructed into a plant expression vector (pCBHUL) and the vector pCBHUL was introduced into rice calli from immature young embryos by particle bombardment. After the calli were regenerated into plantlets on the resistant-selecting media containing hygromycin, they were identified by PCR and Southern blot hybridization. It was indicated that the pi and phb genes were integrated into nucleic genome of the transformed rice plants. GUS activity and the product of the pi gene were determined by GUS staining, Western blot and in situ hybridization at translational level. Eighteen out of 40 plants resistant to hygromycin were positively identified by PCR analysis with the rate of 45%. The pi gene was expressed in 3 out of 18 plants with 17% and 7.5% in 40 plants. The results may provide a clue for exploring whether Rhizobium leguminosarum by. viceae could extend its host range and make the transgenic rice plants have the possibility of being symbiotic, or associative to nitrogen fixation.

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.

大肠埃希菌临床菌株优势β-内酰胺酶基因型及其诱导表达与抑制的研究

目的了解浙江地区大肠埃希菌临床菌株优势β-内酰胺酶基因型及其携带模式、β-内酰胺类抗生素诱导β-内酰胺酶基因表达及组氨酸激酶抑制剂氯氰碘柳胺（CLO）抑制其表达的作用。方法采用微量稀释法和E—test检测大肠埃希菌临床菌株对β-内酰胺类抗生素耐药率和最低抑菌浓度（MIC）。采用PCR及其产物测序法检测大肠埃希菌耐药菌株β-内酰胺酶基因型及携带模式。采用实时荧光定量RT-PCR和β-内酰胺酶确证试验分别检测1／4MIC头孢噻肟或青霉素及CLO对大肠埃希菌耐药菌株6一内酰胺酶基因转录和表达的影响。结果浙江地区61．7％（285／462）大肠埃希菌对青霉素、氨苄青霉素、头孢西丁、头孢噻肟和头孢他啶耐药。285株耐药菌株中，TEM和CTX-M基因检出率（83．2％和75．1％）显著高于KPC、SHV和OXA基因（1．4％～10．2％）（P〈0．01），两种以上β-内酰胺酶基因携带率（68．8％）显著高于单基因（31．2％）（P〈0．01），其中61．4％菌株携带TEM＋CTX—M基因（P〈0．01）。除KPC基因外，1／4MIC头孢噻肟和青霉素能诱导89株β-内酰胺酶单基因菌株TEM、CTX．M、SHV和OXAmRNA水平迅速升高（P〈0．01），但可被50～500μg／ml CLO所抑制（P〈0．01）。100gg／ml CLO预处理后，82．8％～85．6％耐药菌株对上述抗生素敏感（P〈0．01），β-内酰胺酶检出率也从95．1％下降至16．1％（P〈0．01）。结论TEM和CTX．M是浙江地区大肠埃希菌临床菌株优势β-内酰胺酶基因型，并以TEM-1＋CTX．M为优势携带模式。低浓度头孢噻肟和青霉素可经细菌二元信号系统上调β-内酰胺酶基因表达，但可被组氨酸激酶抑制剂CLO所抑制。

Identification of a Lectin Gene Induced in Rice in Response to Magnaporthe grisea Infection

By mRNA differential display, eight induced cDNAs were obtained from rice leaves infected with an incompatible race 131 of Magnaporthe grisea, and one of these cDNAs was highly similar to salt-induced mannose-binding lectin gene. Using this fragment as a probe, a full length cDNA was isolated from a nice cDNA library, which was constructed using mRNA from the incompatible race-infected leaves. Sequence analysis indicates that the cDNA encodes a protein of 15 kD with 145 amino, acids and shares 96% identity at nucleotide level with MRL and salT, but is identical to MRL at amino acid level. Genomic Southern blotting shows that there are two mannose-binding lectin genes in rice genome. Northern blotting analysis indicates that the gene was strongly and specifically induced in rice leaves infected with the incompatible race, suggesting that the lectin induction be involved in the defense of rice to M. grisea.

Cloning and Analysis of CFL—A LFY_like Gene from Cucumber

LFY and LFY _like genes have been shown to control the initiation of floral meristems in higher plants. The homologous cDNA of LFY, CFL, were cloned from cucumber ( Cucumis sativus L.). Southern blot analysis showed it was a single copy gene in the cucumber genome. Northern blot analysis showed that it expressed in the floral buds and young leaves. The possible role of CFL in the floral and vegetative development of cucumber plant was discussed.

Molecular Characterization of Cotton 14-3-3L Gene Preferentially Expressed During Fiber Elongation

The 14-3-3 protein, highly conserved in all eukaryotic cells, is an important regulatory protein. It plays an important role in the growth, amplification, apoptosis, signal transduction, and other crucial life activities of cells. A eDNA encoding a putative 14-3-3 protein was isolated from cotton fiber eDNA library. The eDNA, designated as Gh14-3-3L （Gossypium hirsutum 14-3-3-like）, is 1,029 bp in length （including a 762 bp long open reading frame and 5＇-/3＇-untranslated regions） and deduced a protein with 253 amino acids. The GhI4-3-3L shares higher homology with the known plant 14-3-3 proteins, and possesses the basic structure of 14-3-3 proteins： one dimeric domain, one phosphoralated-serine rich motif, four CC domains, and one EF Hand motif. Northern blotting analysis showed that Gh14-3-3L was predominantly expressed during early fiber development, and reached to the peak of expression in 10 days post anthers （DPA） fiber cells, suggesting that the gene may be involved in regulating fiber elongation. The gene is also expressed at higher level in both ovule and petal, but displays lower or undeteetable level of activity in other tissues of cotton.

Effect of Low Temperature Stress on Sweet Potato S-Adenosyl Methionine Synthetase Gene Expression

[Objective] The mRNA expression level changes of S-adenosylmethionine synthetase （SAMS） under low temperature stress was studied. [Method] Total RNA were extracted from leaves, stem and earthnut of sweet potato 0,12,24,48 and 72 h after low temperature treatement, mRNA expression level was analyzed by reverse expression and Real-time PCR technique. [Result] The expression quality of the gene extracted from leaves, stem and earthnut increased and the expression quality reached the peak point 24,72 and 72 h after low temperature treatment respectively. The expression change of earthnut was the biggest. [Conclusion] Low temperature was good for increasing mRNA expression of relevart genes.

Cloning and Expression of a Profilin Gene from Rapeseed

Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA clone (designated Repro) encoding profilin gene was isolated from rapeseed ( Brassica napus L. cv. canadian Tween) using RT-PCR technique. Sequence analysis showed 82% similarity to Zea mays L. ZmPro3, 85% to Arabidopsis AthPRF1, 82% to Nicotiana tabacum L. NTPRO, 81% to Oryza sativa L. profilin A. A new full-length cDNA was obtained by 5'-RACE and 3'-RACE techniques. Sequence analysis showed that the size of full-length cDNA is 672 bp which contains a major open reading frame of 134 amino, acids, 5' and 3' untranslated regions and a long Poly (A) tail. Northern blot analysis showed that the profilin gene is a pollen and anther specific gene.

Analysis of Differentially Expressed Genes in Genic Male Sterility Cotton(Gossypium hirsutum L.)Using cDNA-AFLP

eDNA amplified fragment length polymorphism （cDNA-AFLP） analysis was used to investigate the differentially expressed genes between sterile and fertile plants of ms5ms6 double-recessive genie male sterility （GMS） two-type line cotton （Gossypium hirsutum L.） at different stages, i.e., sporogenous cell stage, pollen mother cell （PMC） stage, and pollen grain stage. Seventeen differentially expressed fragments were identified. Functional analysis indicated that their corresponding genes may participate in the processes of signal transduction, transcription, energy metabolism, and plant cell wall development. Northern blot demonstrated the credibility of the result of cDNA-AFLE A sterility restorer factor-like gene, which only expressed in fertile anther and was notably homologous to T cytoplasm male sterility restorer factor 2 of maize （Zea mays L.）, was identified in this research.

Cloning and Expression Pattern Analysis of Nitrogen- Starvation-induced Genes in Rice

To understand the regulation system of nitrogen X-starvation in higher plants, a cDNA library from N-starved rice (Oryza sativa L.) seedlings was constructed using rapid subtraction hybridization (RaSH) procedure. Through reverse Northern analysis and Northern blotting, 18 unique known genes and two unique unknown genes were identified, which were up-regulated by N-starvation in rice. The known genes are involved in several metabolisms including carbon metabolism, secondary metabolite synthesis, ubiquitylation and protein degradation, phytohormone metabolism, signal transduction, growth regulator and transcription factors. Different induced expression patterns based on spatial and temporal express ions were found for these genes. The results indicate the cross-talks between N-starvation response and various metabolisms in plants.

Transcriptional Regulation of 10 Mitochondrial Genes in Different Tissues of NCa CMS System in Brassica napus L. and Their Relationship with Sterility

Northern blot analysis was conducted with mitochondrial RNA from seedling leaves, floral buds, and developing seeds of NCa CMS, maintainer line and fertile F1 using ten mitochondrial genes as probes. The results revealed that 9 out of the 10 mitochondrial genes, except for atp6, showed no difference in different tissues of the corresponding materials of NCα CMS system and that they might be constitutively expressed genes. Eight genes, such as orf139, orf222, atpl, cox1, cox2, cob, rm5S, and rm26S, showed no difference among the three tissues of all the materials detected. So the expression of these eight genes was not regulated by nuclear genes and was not tissue-specific. The transcripts of atp9 were identical among different tissues, but diverse among different materials, indicating that transcription of atp9 was neither controlled by nuclear gene nor tissue-specific. Gene atp6 displayed similar transcripts with the same size among different tissues of all the materials but differed in abundance among tissues of corresponding materials and its expression might be tissue-specific under regulation of nuclear gene. Moreover, three transcripts of orf222 were detected in the floral buds of NCa cms and fertile F1, but no transcript was detected in floral buds of the maintainer line.The transcription of orf139 was similar to that of orf222 but only two transcripts of 0.8 kb and 0.6 kb were produced. The atp9 probe detected a single transcript of 0.6 kb in NCa cms and in maintainer line and an additional transcript of 1.2 kb in fertile F1. The relationship of expression of orf222, orf139, and atp9 with NCa sterility was discussed.