Molecular cloning,characterization and promoter analysis of LbgCWIN1 and its expression profiles in response to exogenous sucrose during in vitro bulblet initiation in lily

Lily(Lilium spp.) is an important ornamental flower, which is mainly propagated by bulbs. Cell wall invertases(CWINs), which catalyze the irreversibly conversion of sucrose into glucose and fructose in the extracellular space, are key enzymes participating in sucrose allocation in higher plants. Previous studies have shown that CWINs play an essential role in bulblet initiation process in bulbous crops, but the underlying molecular mechanism remains unclear. Here, a CWIN gene of Lilium brownii var. giganteum(Lbg) was identified and amplified from genomic DNA. Quantitative RT-PCR assays revealed that the expression level of LbgCWIN1 was highly upregulated exactly when the endogenous starch degraded in non-sucrose medium during in vitro bulblet initiation in Lbg. Phylogenetic relationship, motif, and domain analysis of LbgCWIN1 protein and CWINs in other plant species showed that all sequences of these CWIN proteins were highly conserved. The promoter sequence of LbgCWIN1 possessed a number of alpha-amylase-, phytohormone-, light-and stress-responsive cis-elements. Meanwhile, β-glucuronidase(GUS) assay showed that the 459 bp upstream fragment from the translational start site displayed maximal promoter activity. These results revealed that LbgCWIN1 might function in the process of in vitro bulblet initiation and be in the response to degradation of endogenous starch.

Obtaining High Pest_resistant Transgenic Upland Cotton Cultivars Carrying cry1Ac3 Gene Driven by Chimeric OM Promoter

Hypocotyl segments from aseptic seedlings of two important cultivars of upland cotton ( Gossypium hirsutum L.) in Northwest China, 'Xinluzao_1', 'Jinmian_7', 'Jinmian_12' and 'Jihe_321' were transformed respectively by two efficient plant expression plasmids pBinMoBc and pBinoBc via Agrobacterium tumefaciens . In pBinMoBc, cry 1Ac3 gene, which encodes the Bt toxin, is under the control of chimeric OM promoter. In pBinoBc, it is under control of CaMV 35S promoter. After co_cultivation with Agrobacterium tumefimpfaciens LBA4404 (containing pBinMoBc or pBinoBc), kanamycin_resistant selection, somatic embryos were induced and regenerated plants were obtained. Then the regenerated plantlets were grafted to untransformed stocks in greenhouse to produce descendants. The integration of cry 1Ac3 gene and its expression in T 2 generation of transgenic cotton plants were confirmed by Southern hybridization and Western blotting. The analyses of insect bioassay indicated that the transgenic plants of both constructions have significant resistance to the larvae of cotton bollworm ( Heliothis armigera ) and that cry 1Ac3 gene driven by chimeric OM promoter could endue T 2 generation cotton with high pest_resistant ability, implicating that it has a profound application in genetic engineering to breed new pest_resistant cotton varieties.

Cloning and Sequence Analysis of Ca TIP1-1 Gene Promoter from Pepper

In this study, the 1749 bp upstream region of CaTIP1-1 gene was isolated from pepper genome by nested PCR. By using Plant CARE software, it was predicted that the obtained fragment contained typical promoter elements, such as TATA-box, CAAT-box, abiotic stress-related elements and light-response elements.Recombinant plant expression vector PBI121-P_（CaTIP1-1）-GFP was constructed, which was driven by CaTIP1-1 gene promoter and harbored green fluorescent protein reporter gene GFP. After Agrobacterium tumefaciens-mediated transformation with leaf disc method, transgenic tobacco seedlings were obtained successfully. Under a fluorescent microscope, stable green fluorescence signals were observed in T_1 tobacco suspension cell lines. These results suggested that CaTIP1-1 gene promoter could initiate the expression of report gene GFP.This study provided the basis for subsequent application of CaTIP1-1 gene promoter in genetic breeding of plants.

Fe促进的Pt@CeO_(2)催化剂用于CO_(2)加氢制C_(1)产品

在Pt@CeO_(2)核壳纳米球表面引入过渡金属助剂,探究了不同过渡金属的引入对其CO_(2)加氢性能的影响.研究结果表明,Fe物种的引入对加氢性能的提升效果最佳,液体C_(1)产率达到6.34×10^(-2) mmol·g^(-1)_(cat.)·h^(-1).透射电子显微镜(TEM)、X射线衍射(XRD)、X射线光电子能谱(XPS)、N_(2)吸附-脱附实验、CO_(2)程序升温脱附(CO_(2)-TPD)和H_(2)程序升温还原(H_(2)-TPR)等表征结果表明,Fe物种在Pt@CeO_(2)表面均匀分散,且Fe的存在降低了Pt物种的电荷密度,产生了更多的Pt^(2+)物种,提高了产物中甲醇的选择性.此外,Fe的存在还促进了更多氧空位(O_(v))的形成,进而促进了对CO_(2)的吸附及后续的加氢反应,提高了催化活性.

基于BP神经网络模型优化Fe_(1-x)O基氨合成催化剂

运用BP神经网络建立了助催化剂含量与催化剂活性之间的预测模型,对Fe_(1-x)O基氨合成催化剂的助催化剂进行优化。首先将前期实验数据整理归纳为含有3、4、5、6和7个助催化剂等5类催化剂,以助催化剂含量(体积分数)为输入变量,以425℃反应器出口氨浓度(活性)为输出变量,对助催化剂进行优化。结果表明,BP神经网络预测模型拟合值均方误差最高为0.2784,预测值均方误差最高为0.1592,构建的BP神经网络模型准确度较高。在该模型的基础上,运用多种群遗传算法进行极值寻优,求解最优的催化剂配方,并进行实验验证。结果表明,根据优化结果制备5个样品的实验测定值与预测值的相对误差最高为2.88%,优化结果较为准确;含有7个助催化剂的催化剂活性最高为18.83%,比原样本的统计平均活性值(17.52%)高1.31%,相对提高7.48%,助催化剂含量优化取得满意的结果。

Underexpression of LATS1 TSG in colorectal cancer is associated with promoter hypermethylation

AIM:To investigate large tumor suppressor 1 (LATS1 ) expression, promoter hypermethylation, and microsatellite instability in colorectal cancer (CRC).METHODS:RNA was isolated from tumor tissue of 142 CRC patients and 40 colon mucosal biopsies of healthy controls. After reverse transcription, quantitative polymerase chain reaction (PCR) was performed, and LATS1 expression was normalized to expression of the ACTB and RPL32 housekeeping genes. To analyze hypermethylation, genomic DNA was isolated from 44 tumor CRC biopsies, and methylation-specific PCR was performed. Microsatellite instability (MSI) status was checked with PCR using BAT26, BAT25, and BAT40 markers in the genomic DNA of 84 CRC patients, followed by denaturing gel electrophoresis. RESULTS:Decreased LATS1 expression was found in 127/142 (89.4%) CRC cases with the average ratio of the LATS1 level 10.33 ± 32.64 in CRC patients vs 32.85 ± 33.56 in healthy controls. The lowest expression was found in Dukes' B stage tumors and G1 (welldifferentiated) cells. Hypermethylation of the LATS1 promoter was present in 25/44 (57%) CRC cases analyzed. LATS1 promoter hypermethylation was strongly associated with decreased gene expression; methylated cases showed 162× lower expression of LATS1 than unmethylated cases. Although high-grade MSI (mutation in all three markers) was found in 14/84 (17%) cases and low-grade MSI (mutation in 1-2 markers) was found in 30/84 (36%) cases, we found no association with LATS1 expression. CONCLUSION:Decreased expression of LATS1 in CRC was associated with promoter hypermethylation, but not MSI status. Such reduced expression may promote progression of CRC.

Promoter methylation and mRNA expression of DKK-3 and WIF-1 in hepatocellular carcinoma

AIM:To investigate the promoter methylation status and mRNA expression of DKK-3 and WIF-1 gene in hepatocellular carcinoma(HCC).METHODS:DKK-3 and WIF-1 acted as Wnt-antagonists and tumor suppressors,but hypermethylation of the gene promoter and low mRNA expression activated Wnt signaling aberrantly and induced the development of HCC.Methylation status of the DKK-3 and WIF-1 gene promoter was investigated using methylation specific polymerase chain reaction(PCR) in tumor and adjacent non-cancerous tissues from 33 HCC patients and 20 normal liver tissues served as control.The expression of DKK-3 and WIF-1 mRNA was also determined by real-time quantitative reverse transcriptase PCR.The relationship between methylation,mRNA expression,and clinical data,as well as methylation and mRNA expression of the two genes were analyzed.RESULTS:The methylation of DKK-3 and WIF-1 genes in HCC increased significantly compared with adjacent non-cancerous tissues and normal control tissues(χ2 =7.79,P < 0.05;χ2 = 4.89,P < 0.05),and no significant difference in methylation between adjacent non-cancerous tissues and normal control tissues was observed.In HCC tissues,significant differences in the DKK-3 promoter methylation were observed in age and cirrhosis,and significant differences of the WIF-1 promoter methylation were observed in HBsAg and cirrhosis.The average expression of DKK-3 mRNA in HCC and adjacent non-cancerous tissues was increased significantly compared with normal control tissues.The average expression of WIF-1 mRNA showed no significant difference among the three tissues.The mRNA expression of DKK-3 gene in HCC was decreased as the pathological grade increased.CONCLUSION:The aberrant promoter methylation and decreased expression of DKK-3 and WIF-1 may be an important mechanism in HCC,and may be a far-reaching significance in early diagnosis and therapy of HCC.

Promoter hypomethylation and reactivation of MAGE-A1 and MAGE-A3 genes in colorectal cancer cell lines and cancer tissues

AIM： To verify the expression and methylation status of the MAGE-A1 and MAGE-A3 genes in colorectal cancer tissues and cancer cell lines. METHODS： We evaluated promoter demethylation status of the MAGE-A1 and MAGE-A3 genes by RT-PCR analysis and methylation-specific PCR （MS-PCR）, as well as sequencing analysis, after sodium bisulfite modification in 32 colorectal cancer cell lines and 87 cancer tissues. RESULTS： Of the 32 cell lines, MAGE-A1 and MAGE-A3 expressions were observed in 59% and 66%, respectively. Subsequent to sodium bisulfite modification and MSPCR analysis, the promoter hypomethylation of MAGE-A1 and MAGE-A3 was confirmed in both at 81% each. Promoter hypomethylation of MAGE-A1 and MAGE-A3 in colorectal cancer tissues was observed in 43% and 77%, respectively. Hypomethylation of MAGE-A1 and MAGE-A3 genes in corresponding normal tissues were observed in 2% and 6%, respectively. CONCLUSION： The promoter hypomethylation of MAGE genes up-regulates its expression in colorectal carcinomas as well as in gastric cancers and might play a significant role in the development and progression of human colorectal carcinomas.

Promoter methylation status of hMLH1,MGMT,and CDKN2A/p16 in colorectal adenomas

AIM:To investigate aberrant DNA methylation of CpG islands and subsequent low-or high-level DNA microsatellite instability(MSI)which is assumed to drive colon carcinogenesis. METHODS:DNA of healthy individuals,adenoma(tu-bular or villous/tubulovillous)patients,and colorectal carcinoma patients who underwent colonoscopy was used for assessing the prevalence of aberrant DNA methylation of human DNA mismatch repair gene mutator L homologue 1(hMLH1),Cyclin-dependent kinase inhibitor 2A(CDKN2A/p16),and O-6-methylguanine DNA methyltransferase(MGMT),as well as their rela- tion to MSI. RESULTS:The frequency of promoter methylation for each locus increased in the sequence healthy tissue/adenoma/carcinoma.MGMT showed the highest frequency in each group.MGMT and CDKN2A/p16 presented a statistically significant increase in promoter methylation between the less and more tumorigenic forms of colorectal adenomas(tubular vs tubullovillous and villous adenomas).All patients with tubulovillous/villous adenomas,as well as all colorectal cancer patients,showed promoter methylation in at least one of the examined loci.These findings suggest a potentially crucial role for methylation in the polyp/adenoma to cancer progres- sion in colorectal carcinogenesis.MSI and methylation seem to be interdependent,as simultaneous hMLH1, CDKN2A/p16,and MGMT promoter methylation was present in 8/9 colorectal cancer patients showing the MSI phenotype. CONCLUSION:Methylation analysis of hMLH1,CD- KN2A/p16,and MGMT revealed specific methylation profiles for tubular adenomas,tubulovillous/villous adenomas,and colorectal cancers,supporting the use of these alterations in assessment of colorectal tumorigenesis.

A novel SATB1 binding site in the BCL2 promoter region possesses transcriptional regulatory function

BCL2 is a key regulator of apoptosis.Our previous work has demonstrated that special AT-rich sequence-binding protein 1 （SATB1） is positively correlated with BCL2 expression.In the present study,we report a new SATB1 binding site located between P1 and P2 promoters of the BCL2 gene.The candidate SATB1 binding sequence predicted by bioinformatic analysis was investigated in vitro and in vivo by electrophoretic gel mobility shift assays （EMSA） and chromatin immunoprecipitation （ChIP）.One 25-bp sequence,named SB1,was confirmed to be SATB1 binding site.The regulatory function of SB1 and its relevance to SATB1 were further examed with dual-luciferase reporter assay system in Jurkat cells.We found that SB1 could negatively regulate reporter gene activity.Mutation of SATB1 binding site further repressed the activity.Knockdown of SATB1 also enhanced this negative effect of SB1.Our data indicate that the SB1 sequence possesses negative transcriptional regulatory function and this function can be antagonized by SATB1.

Functional analysis of two Sp1/Sp3 binding sites in murine Nanog gene promoter

Nanog gene plays a key role in maintaining pluripotency of ES cells and early embryonic cells. A 5＇ flank sequence of the Nanog gene has been reported to be regulated differentially, and two regulatory elements within the Nanog promoter, namely Oct-4 and Sox-2 binding sites, have been identified to regulate the transcriptional activity ofNanog gene. In this report, we identified the role of two putative Spl binding sites located in the Nanog gene 5＇-flanking region in regulation ofmurine Nanog gene transcription. Mutation studies showed that the two sites were essential for the Nanog promoter activity. Gel shift and supershift analysis showed that both sites specifically bind Spl and Sp3. Furthermore, overexpression of dominant-negative Spl or Sp3 mutants significantly inhibits Nanog promoter activity. These results suggest that the transcription factor Spl and Sp3 are important for Murine Nanog gene expression.

Myeloid zinc finger 1(MZF1) is the most important transcriptional factor for porcine follistatin promoter

Follistatin （FS） is a secreted protein, which was originally isolated from porcine follicular fluid. Expression of follistatin is tightly regulated during porcine growth and development. To study the essential transcriptional regions of the porcine FS promoter, ten primer pairs were designed to amplify segments with different lengths of the FS promoter from -1 800 to ＋16 bp. The products were then inserted into the pGL3-basic vector to analyze the relative luciferase activity. The results showed that the most remarkable changes of promoter activity were observed between constructs （-302/＋16 bp）-FS and （-180/＋16 bp）-FS （P〈0.01）. Further research showed that the reconstructed reporter plasmid lacking myeloid zinc finger 1 （MZF1） binding sequence had significantly decreased luciferase activity （P〈0.05）. Furthermore, the FS protein expression was significantly increased in PK15 cells while the MZF1 was overexpressed, suggesting that the short sequence ＂TCCCCACC＂ （the recognition site of transcription factor MZF1） was the most important for FS transcription activation in the porcine.

Promoter polymorphism of transforming growth factor-β1 gene and ulcerative colitis

AIM: To elucidate the possible difference in two promoter polymorphisms of the transforming growth factor-β1 (TGF-β1) gene (-800G > A, -509C > T) between ulcerative colitis (UC) patients and normal subjects.METHODS: A total of 155 patients with established ulcerative colitis and 139 normal subjects were selected as controls. Two single nucleotide polymorphisms within the promoter region of TGF-β1 gene (-509C > T and -800G > A) were genotyped using PCR-RFLP. RESULTS: There was a statistically significant difference in genotype and allele frequency distributions between UC patients and controls for the -800G > A polymorphism of the TGF-β1 gene (P < 0.05). The frequency of the TGF-β1 gene polymorphism at position -800 showed that the AA genotype and the allele A frequencies significantly differed between the patients and healthy controls (P < 0.05). At position -509, there was no statically significant difference in genotype and allele frequency between the patients and control subjects.CONCLUSION: The results of our study indicate that there is a significant difference in both allele and genotype frequency at position -800G > A of TGF-β1 gene promoter between Iranian patients with UC and normal subjects.

Dual-targeting AAV9P1-mediated neuronal reprogramming in a mouse model of traumatic brain injury

Traumatic brain injury results in neuronal loss and glial scar formation.Replenishing neurons and eliminating the consequences of glial scar formation are essential for treating traumatic brain injury.Neuronal reprogramming is a promising strategy to convert glial scars to neural tissue.However,previous studies have reported inconsistent results.In this study,an AAV9P1 vector incorporating an astrocyte-targeting P1 peptide and glial fibrillary acidic protein promoter was used to achieve dual-targeting of astrocytes and the glial scar while minimizing off-target effects.The results demonstrate that AAV9P1 provides high selectivity of astrocytes and reactive astrocytes.Moreover,neuronal reprogramming was induced by downregulating the polypyrimidine tract-binding protein 1 gene via systemic administration of AAV9P1 in a mouse model of traumatic brain injury.In summary,this approach provides an improved gene delivery vehicle to study neuronal programming and evidence of its applications for traumatic brain injury.

山羊NPC1基因核心启动子鉴定与转录调控分析

NPC1基因编码的C型尼曼匹克蛋白1(NPC1)参与脂筏形成、病原微生物内吞以及内吞体的胞内转运等过程。本试验旨在阐明海南黑山羊NPC1基因的转录调控机制,为研究病原微生物与宿主细胞互作提供理论参考。首先,以山羊NPC1基因1466 bp启动子序列为模板,构建9个启动子5'端连续缺失的双荧光素酶报告载体,筛选NPC1基因核心启动子区;继而对核心启动子区的关键转录因子进行预测分析,构建转录因子结合位点缺失的双荧光素酶报告载体,筛选NPC1基因的关键转录因子结合位点。结果表明:山羊NPC1基因的核心启动子区位于转录起始位点上游-195 bp至-60 bp,并且该区域存在E2F3(-134/-120 bp)、SREBP-1(-107/-96 bp)、SP1(-68/-62 bp)等多个转录因子结合位点。双荧光素酶报告实验结果表明,缺失E2F3、SREBP-1、SP1三个转录因子结合位点均会使山羊NPC1基因启动子的转录活性显著下降(P<0.01),且缺失SREBP-1结合位点后NPC1基因转录活性下降最显著。提示,SREBP-1转录因子对海南黑山羊NPC1基因转录活性具有重要的调控作用。本试验成功鉴定山羊NPC1基因的核心启动子区(-195/-60 bp)及该区域的关键转录因子结合位点SREBP-1,为进一步研究山羊NPC1基因介导病原微生物与宿主互作分子机制提供理论基础。

Identification and functional characterization of the MdHB-1 gene promoter sequence from Malus×domestica

Homeobox 1 in Malusxdomestica （MdHB-1） is a transcription factor that belongs to homeodomain-leucine zipper I （HD-Zip I） protein subfamily. According to previous reports, MdHB-1 could regulate ethylene synthesis by binding with the MdAC01 promoter, but other functions of MdHB-1 are still unknown. To reveal more clues concerning the characters of the MdHB-1 gene promoter and the functions of MdHB-1, the promoter region of MdHB-1 was cloned from the Royal Gala apple genome and recombined with the 13-glucuronidase （GUS） gene in this study. This research was conducted in Nicotiana tabacum and supported by Agrobacterium-mediated transient transformation and bioinformatics analysis. Deletion analysis of the MdHB-1 promoter showed that the GUS gene could be activated by serially deleted promoters, and the activity promoted by 680 nucleotides （nt） was the lowest. The region, which is 266 nt upstream of the initiation code （ATG）, was effective for GUS expression. Meanwhile, the activity of the MdHB-1 promoter （-1 057 nt）, which was stronger than MdHB-1 promoter （-1 057 to -266 nt） and lack the 5＂-untranslated region （5＂-UTR）, showed that 5＂-UTR may have a positive effect on gene transcription. After the sequence analysis, the cis-acting elements that respond to hormones and environmental stresses were identified in the promoter region. The MdHB-1 promoter （1 057 nt） activity in Nicotiana tabacum was positively induced by ethrel and darkness, and it was suppressed by gibberellic acid （GA）, whereas abscisic acid （ABA）, salicylic acid （SA）, wounding, and Pseudomonas syringae pv. tomato （DC3000） treatments revealed a slight auxo-action. These results reveal that the MdHB-1 promoter receive internal or external signals, and MdHB-1 may refer to many biological activities in apple, such as its stress response, development, and ripening.

Sp1 contributes to overexpression of stanniocalcin 2 through regulation of promoter activity in colon adenocarcinoma

BACKGROUND Aberrant expression of stanniocalcin 2 (STC2) is implicated in colon adenocarcinoma (COAD). A previous study identified that STC2 functions as a tumor promoter to drive development of some cancers, but the role of its overexpression in the development of COAD remains unclear. AIM To evaluate the regulation mechanism of STC2 overexpression in COAD. METHODS The expression of STC2 in COAD was assessed by TCGA COAD database and GEO (GSE50760). Methylation level of the STC2 promoter was evaluated with beta value in UALCAN platform, and the correlation between STC2 expression and survival rate was investigated with TCGA COAD. Transcription binding site prediction was conducted by TRANSFAC and LASAGNA, and a luciferase reporter system was used to identify STC2 promoter activity in several cell lines, including HEK293T, NCM460, HT29, SW480, and HCT116. Western blotting was performed to evaluate the role of Sp1 on the expression of STC2. RESULTS The central finding of this work is that STC2 is overexpressed in COAD tissues and positively correlated with poor prognosis. Importantly, the binding site of the transcription factor Sp1 is widely located in the promoter region of STC2. A luciferase reporter system was successfully constructed to analyze the transcription activity of STC2, and knocking down the expression of Sp1 significantly inhibited the transcription activity of STC2. Furthermore, inhibition of Sp1 remarkably decreased protein levels of STC2. CONCLUSION Our data provide evidence that the transcription factor Sp1 is essential for the overexpression of STC2 in COAD through activation of promoter activity. Taken together, our finding provides new insights into the mechanism of oncogenic function of COAD by STC2.

Study on RIZ1 gene promoter methylation status in human esophageal squamous cell carcinoma

AIM： To investigate the promoter region methylation status of retinoblastoma protein-interacting zinc finger gene 1 （RIZ1） in the human esophageal squamous cell carcinoma （ESCC） cell lines and tissues and verify the relationship between methylation of RIZ1 and oncogen- esis, tumor progression and metastasis etc of ESCC. METHODS： Methylation-specific polymerase chain reac- tion （MSP） was used to investigate the promoter region methylation status of RIZ1 in 6 ESCC cell lines. One cell line where RIZ1 promoter region methylation was de- tected was selected for the next study, where the cell line was treated with 5-aza-CdR. Real-time polymerase chain reaction was used to investigate its influence on the transcription of RIZ1. Experiments using frozenpathological specimens from 47 ESCC patients were performed using the same MSP methodology. RESULTS： Promoter methylation of RIZ1 gene was detected in TEl3, CaEs17 and EC109 cell lines and the cell line TEl3 was chosen for further study. The expression of RIZl mRNA in TE-13 was up-regulated after treatment with 5-aza-CdR. The rate of methyla- tion in carcinomas tissues was significantly higher than those in matched neighboring normal and distal ending normal tissue, and the deviation of data was statisti- cally significant （2,2 = 24.136, P 〈 0.01）. Analysis of the gender, age familial history, tumour deviation, tumour saturation, lymph gland displacement and clinical stag- ing of 47 samples from ESCC patients showed that the fluctuation of data was not statistically significant. CONCLUSION： Promoter methylation may play an im- portant role in the epigenetic silencing of RIZ1 gene expression in human ESCC. RIZ1 is considered to be a potential tumor suppressor gene and may be a biologi- cal parameter for testing early stage human ESCC.

基于Nrf2/HO-1启动子筛选抗水生病毒药物的方法

为建立基于Nrf2、HO-1启动子活性的抗病毒药物筛选方法,实验以胖头鱥上皮细胞系(FHM)为材料,构建Nrf2、HO-1启动子重组质粒,利用双荧光素酶报告系统检测不同浓度(0、3.1、6.3、12.5、25.0、50.0μg/mL)的白藜芦醇、水飞蓟宾、穿心莲内酯及姜黄素对启动子活性的影响,并利用鲤春病毒血症病毒(spring viremia of carp virus,SVCV)和蛙虹彩病毒(rana grylio iridovirus,RGV)验证阳性药物的抗病毒效果。结果显示,Nrf2、HO-1启动子序列中存在FOX家族、IRF家族等多种转录因子的结合位点,并分别存在1个和3个与甲基化相关的CpG岛。双荧光素酶报告实验显示,水飞蓟宾、穿心莲内酯及姜黄素可激活Nrf2、HO-1启动子。药物浓度梯度实验显示,6.3μg/mL的姜黄素和穿心莲内酯可显著上调Nrf2和HO-1的启动子活性。病毒感染后的细胞病变效应、病毒的复制及病毒滴度结果均表明姜黄素和穿心莲内酯可抑制SVCV和RGV的感染。综上,本实验建立的pGL3-Nrf2和pGL3-HO-1启动子报告质粒,可用于抗水生病毒药物的筛选,也为Nrf2、HO-1转录调控机制的研究提供了工具。

Functional analysis of the promoter of the dmrt1 gene in Chinese tongue sole,Cynoglossus semilaevis

The dmrt1 (doublesex and mab-3 related transcription factor 1) gene is considered to play an essential role in testis differentiation and development among metazoan species. As an economically important marine fish in China, Chinese tongue sole (Cynoglossus semilaevis) possess a ZW/ZZ sex-determining system, and the females usually grow two to four times bigger than males. Previous studies have confirmed that Z-linked dmrt1 gene is the male determining gene in C. semilaevis and TALENs-mediated knocking-out individuals showed increased growth phenotype. In order to investigate the function of dmrt1 regulatory regions and its potential application, we have cloned the promoter of C. semilaevis dmrt1. The acquired sequence consisted of a 5'-upstream sequence and a partial exon1;functional analysis showed that there were two positive regulatory regions in the promoter, as well as one negative region and one neutral region. These positive regions were then combined to obtain several newly constructed promoters. Both in vitro and in vivo analysis confirmed one of them (dmrt1-Δ3+Δ4) showed elevated promoting activity compared to the full-length promoter and could drive exogenous gene expression in tongue sole gonads. Our results will provide useful information for understanding the regulatory mechanism of dmrt1 gene during C. semilaevis sex determination, and the endogenous promoter will facilitate the transgenic research for analyzing sex- and growth-related genes.