苹果乙烯响应因子基因MdERF11对脱落酸的敏感性分析

以‘嘎拉’苹果(Malus×domestica Borkh.)为试材,克隆了乙烯响应因子基因MdERF11(序列号MDP0000756341)。测序发现,该基因包含全长为483 bp的完整开放阅读框,编码161个氨基酸。系统进化树分析表明,这一乙烯响应因子与拟南芥AtERF11蛋白同源序列相似性最高。利用PlantCare数据库进行基因启动子顺式作用元件预测,MdERF11启动子序列中含有与脱落酸(ABA)、乙烯及干旱信号相关的顺式作用元件。荧光定量PCR分析表明,MdERF11在苹果的各组织中均有表达,在叶柄和果实中表达量相对较高;并且MdERF11的表达明显受到ABA的诱导。在外源ABA的处理下,MdERF11过量表达的苹果愈伤组织的生长势明显比野生型强,表明MdERF11降低了苹果愈伤组织对ABA的敏感性。     

苹果细胞分裂素响应因子基因MdCRF4的分离与功能鉴定

以‘嘎拉’苹果(Malus × domestica‘Royal Gala’)为研究材料,利用同源克隆和PCR技术,分离了苹果细胞分裂素响应因子(cytokinin response factor)基因MdCRF4。该基因开放阅读框(ORF)为1077 bp,编码含有358个氨基酸的蛋白。保守结构域和系统进化树分析显示,MdCRF4蛋白包含一个保守的CRF结构域和一个保守的AP2/ERF结构域,与梨PbCRF4同源性最高。基因表达分析显示,该基因主要在苹果根和叶中表达并且响应细胞分裂素。EMSA试验显示MdCRF4原核表达蛋白能够绑定DRE(ACCGAC)序列。在‘王林’苹果愈伤组织中超表达MdCRF4,其花青苷积累显著增加,表明MdCRF4在调控花青苷积累中发挥重要作用。     

苹果乙烯响应因子MdERF1B-like的克隆与功能鉴定

以‘泰山早霞’苹果发育期中未着色和着色的果实为试材,利用q RT-PCR分析花青苷和乙烯通路相关基因的表达。分离出1个乙烯响应因子,暂命名为MdERF1B-like(MDP0000167207),其开放阅读框为690bp,编码229个氨基酸。在‘泰山早霞’苹果着色与未着色果实中,MdERF1B-like表达量变化与花青苷合成基因MdDFR、MdANS和调控基因MdMYB9的表达趋势一致。系统进化树分析表明,MdERF1B-like位于第Ⅸ组,与PyERF1B-like亲缘关系最近。在‘王林’苹果愈伤组织中过表达MdERF1B-like,其花青苷含量显著高于对照。分析MdMYB9启动子序列,其长度为746 bp,序列中含有1个ERF潜在结合元件RAA(TGTTG),酵母单杂表明MdERF1B-like与MdMYB9启动子结合。进一步通过荧光素酶报告试验验证MdERF1B-like促进MdMYB9启动子的转录活性。因此,MdERF1B-like可能通过对MdMYB9的调控来促进苹果花青苷积累。     

钙对“新红星”苹果乙烯生成的作用

本研究采用减压渗钙的方法处理‘新红星’苹果,结果表明,钙处理降低了果实乙烯含量,并降低果实ACC含量与EFE活性,但增大果实MACC含量,还影响了果实蛋氨酸含量。     

苹果WRKY转录因子家族基因生物信息学分析

 利用生物信息学方法对苹果MdWRKY转录因子家族成员、基因分类、染色体定位、系统进化关系和结构域序列保守性进行了预测,并分析基因在果实成熟期和砧穗互作中的表达差异。苹果MdWRKY家族包含116个基因,分为GroupⅠ、GroupⅡ和GroupⅢ,其中GroupⅡ又可细分为GroupⅡa、GroupⅡb、GroupⅡc、GroupⅡd和GroupⅡe亚类;苹果的17条染色体均有WRKY转录因子分布,其中第1条染色体上的分布最多,有12个WRKY基因分布。MdWRKY编码的蛋白在118 ~ 965个氨基酸范围内,等电点位于4.81 ~ 10.16之间。Microarray分析发现,在苹果果实成熟时期和砧木接穗互作过程中,多数MdWRKY基因的表达都有不同程度的变化。     

苹果乙烯响应因子MdERF3促进花青苷和原花青苷积累

以'嘎拉'苹果(Malus×domestica'Royal Gala')为材料,克隆乙烯响应因子(Ethylene Response Factor 3)基因MdERF3。构建酵母载体pGBKT7-MdERF3和原核表达载体PET32a-MdERF3,酵母转化试验表明,MdERF3转录因子具有转录激活活性。电泳迁移率试验分析显示,MdERF3-HIS原核诱导蛋白能够直接结合GCC和DRE序列。构建pCAMBIA1300-MdERF3超表达载体,转化苹果愈伤组织和拟南芥植株,并瞬时转化苹果叶片,转基因材料花青苷和原花青苷积累显著高于野生型。以上结果表明MdERF3转录因子具有转录激活活性以及对GCC和DRE序列的结合能力;MdERF3在调控花青苷和原花青苷积累过程中发挥重要作用。     

外源钙对苹果果实乙烯生成的影响

 外源Ca2 + 浓度低于350 mmol ·L^-1时, 可促进‘红富士’苹果果实乙烯释放, 在400 ～1 000 mmol·L^-1之间时, 乙烯的释放速率仅为对照的29. 8 %～41 %。分别用高浓度(500 mmol·L^-1) 与低浓度(80 mmol·L^-1) CaCl₂处理果实12 h, 随处理时间延长, 低浓度钙促进乙烯释放量持续增加, 12 h 时乙烯释放量是2 h 时的2. 9 倍, 而高浓度钙抑制乙烯生成的效应比较稳定, 保持在12. 6～14. 4 nL·g^-1。低浓度的Mg²⁺、Mn²⁺也有刺激乙烯生成的作用。Ca²⁺的胞内流动抑制剂La³⁺及CaM的抑制剂TFP (三氟啦嗪) 在80 mmol·L^-1的CaCl₂存在时, 明显抑制乙烯释放。     

Effect of circRNA_000203 on fibrotic phenotypes in mouse cardiac fibroblasts

AIM: To determine circular RNA (circRNA) profiles in the diabetic mouse myocardium, and to investigate the effect of circRNA_000203 on fibrotic phenotypes in cardiac fibroblasts.METHODS: Masson trichrome staining was performed on the myocardium of the diabetic db/db mice and the non diabetic db/m control mice. circRNA expression profile in the diabetic myocardium was detected by circRNAs microarray. The expression of circRNA_000203 was determined by real time fluorescence quantitative PCR (RT-qPCR). Recombinant circRNA_000203 adenovirus was prepared for enforced the expression of circRNA_000203 in mouse cardiac fibroblasts. The expression of Col1a2, Col3a1and α-SMA was determined in circRNA_000203-modified cardiac fibroblasts, respectively. RESULTS: Masson trichrome staining showed that fibrosis was increased in the diabetic mouse myocardium. The results of circRNA array detection revealed that circRNAs were dysregulated in the diabetic myocardium. circRNA_000203 was up-regulated in the diabetic myocardium. Significant over-expression of circRNA_000203 was achieved in the cardiac fibroblasts after infection with the recombinant circRNA_000203 adenovirus. The mRNA and protein expression of Col1a2, Col3a1 and α-SMA was significantly increased in the cardiac fibroblasts with over-expression of circRNA_000203.CONCLUSION: circRNA_000203 is up-regulated in the diabetic mouse myocardium. It has pro-fibrotic effect on the cardiac fibroblasts.     

miR-381-3p protects hippocampal neurons of depressive rats from injury via BDNF

AIM: To investigate the effect of microRNA (miR)-381-3p on neuronal injury in the hippocampus of depressive rats and the possible regulatory mechanism.METHODS: The rat model of depression was established by subcutaneous injection of corticosterone and the model rats received fluoxetine treatment. The body weight, open field test and biochemical indexes were measured for judging the therapeutic effect of fluoxetine. The expression of miR-381-3p, brain-derived neurotrophic factor (BNDF), Bcl-2 and Bax in the hippocampus was determined. The cultured neonatal rat hippocampal neurons were pre-transfected with miR-381-3p inhibitor and then incubated with corticosterone. The cell viability, and the expression of miR-381-3p, BNDF, Bcl-2 and Bax were detected. The targeted regulatory role of miR-381-3p in BDNF was verified by luciferase reporter assay.RESULTS: Compared with depression group, fluoxetine increased the body weight, the number of cross-field activities and the content of norepinephrine, inhibited the expression of miR-381-3p and promoted the expression of BNDF in the hippocampus. miR-381-3p silencing reversed the effect of corticosterone, resulting in the increase in the survival rate of hippocampal neurons, upregulation of BDNF and Bcl-2, and downregulation of Bax expression. miR-381-3p targeted the regulation of BNDF expression.CONCLUSION: Silencing miR-381-3p protects rat hippocampal neurons from corticosterone injury, and its mechanism may be related to the upregulation of BNDF expression.     

Expression of miR-625-3p in colorectal carcinoma tissues and cells

AIM: To investigate the expression of microRNA-625-3p (miR-625-3p) in colorectal carcinoma (CRC) and its underlying mechanism. METHODS: Quantitative real-time PCR was employed to detect the levels of miR-625-3p expression in different CRC cell lines, CRC tissues and pair-matched adjacent normal tissues. The relationships between the expression levels of miR-625-3p and the patients' clinicopathological parameters were estimated. The effects of miR-625-3p on the apoptosis and the cell mitotic cycle of CRC cells were analyzed with propidium iodide staining and flow cytometry. The effect of miR-625-3p on the apoptosis-related proteins was analyzed by Western blot. RESULTS: The expression level of miR-625-3p in the CRC tissues was higher than that in the pair-matched adjacent normal tissues (P<0.05). The expression of miR-625-3p in the CRC tumor tissues was significantly correlated with the tumor infiltrative depth, TNM stage and distant metastasis (P<0.05). The expression levels of miR-625-3p in CRC SW620 cells were higher than that in SW480 cells. The CRC cell mitotic cycle was significantly inhibited and cell apoptosis was significantly promoted when the expression of miR-625-3p was inhibited (P<0.05). The expression of Bax protein didn't change and the expression of Bcl-2 protein increased after miR-625-3p mimics were transfected into CRC SW620 cells(P<0.05). CONCLUSION: miR-625-3p may be a promising approach for the treatment of CRC by promoting cell proliferation and inhibiting apoptosis.     

Expression and effect of miR-139-3p in apoptotic neonatal rat cardiomyocytes induced by hypoxia

AIM:To investigate the expression and clinical significance of microRNA-139-3p (miR-139-3p) in the apoptosis model of cardiomyocytes induced by hypoxia. METHODS:Under normal and hypoxic conditions, the expression of miR-139-3p in neonatal rat cardiomyocytes was detected by RT-qPCR. miR-139-3p inhibitor and miR-139-3p inhibitor negative control were transfected into the primary neonatal rat cardiomyocytes. The transfected cardiomyocytes were cultured in closed anoxic box (95% N₂ and 5% CO₂) at 37℃ for 12 h. Flow cytometry and Western blot were used to determine the apoptosis of cardiomyocytes. RESULTS:After hypoxia for 12 h, the expression level of miR-139-3p and the apoptotic rate of the cardiomyocytes in hypoxia group were significantly higher than those in normal group (P<0.05). Moreover, compared with the miR-139-3p inhibitor negative control group, the apoptotic rate of the cardiomyocytes was significantly decreased in miR-139-3p inhibitor group (P<0.05). CONCLUSION:The expression of miR-139-3p is signi-ficantly increased in apoptotic neonatal rat cardiomyocytes induced by hypoxia. Inhibition of miR-139-3p expression reduces hypoxia-induced apoptosis of cardiomyocytes.     

miR-140-3p inhibits viability,invasion and migration of non-small-cell lung cancer A549 cells by targeting PD-L1

AIM: To explore the target relationship between microRNA-140-3p (miR-140-3p) and programmed cell death ligand 1 (PD-L1) and their effect on the viability, migration and invasion of non-small-cell lung cancer A549 cells.METHODS: RT-qPCR was used to detect the miR-140-3p expression in HLF-1, A549 and H1299 cells, and then the A549 cells with the most significant difference were selected as the subsequent research object. TargetScan software and dual-luciferase reporter assay were performed to predict and confirm the target relationship between miR-140-3p and PD-L1. RT-qPCR and Western blot were used to determine the effects of miR-140-3p mimic and inhibitor on PD-L1 expression level. MTT assay was used to detect the viability of A549 cells. Transwell assay was performed to detect the migration and invasion abilities of the A549 cells.RESULTS: miR-140-3p was significantly down-regulated in the A549 cells and H1299 cells (P<0.05). Transfection with miR-140-3p mimic decreased the expression of PD-L1 and inhibited the viability, migration and invasion of the A549 cells. Transfection with pcDNA3.0-PD-L1 reversed the inhibitory effect of miR-140-3p on the viability, migration and invasion of the A549 cells.CONCLUSION: miR-140-3p inhibits the viability, migration and invasion of A549 cells by targeting PD-L1.