茶树蛋白激酶基因CsCIPK的克隆与表达特性分析

以茶树品种‘龙井43’作为材料,利用RT-PCR方法,从茶树的cDNA中克隆得到1个编码蛋白激酶的基因,命名为CsCIPK。序列分析表明,CsCIPK开放阅读框长度为1 341 bp,编码446个氨基酸,蛋白质分子量为414234。蛋白功能域预测和多重对比显示,CsCIPK蛋白含有1个保守的N端激酶结构域和1个相对不保守的C端调节结构域,即丝氨酸/苏氨酸激酶结构域和NAF结构域。理化性质、亲/疏水性、无序化分析显示,CsCIPK属于疏水性蛋白,理论等电点为7.04,有4段无序化区域,其二级结构分析显示主要由α螺旋、不规则卷曲组成。通过实时荧光定量PCR对‘龙井43’和‘安吉白茶’中的CsCIPK表达特性进行分析。结果显示‘龙井43’中CsCIPK的相对表达量在高温、干旱及盐处理4 h、低温处理24 h时达到最高。‘安吉白茶’中CsCIPK的相对表达量在高温及盐处理4 h、低温及干旱处理1h时达到最高。CsCIPK在‘龙井43’的根中,‘安吉白茶’茎中表达量最高。不同浓度的GA和IBA处理‘龙井43’茶苗,结果显示0.2 mmol·L-1 GA处理后,CsCIPK表达量先升高后下降,6 d时处理组为对照组的62倍;0.6 mmol·L-1 IBA处理后,CsCIPK的表达量在3 d时显著高于对照组;不同浓度GA和IBA处理后,9 d时CsCIPK表达量均显著低于对照。     

采用胚挽救获得Ogura CMS甘蓝与甘蓝型油菜的种间杂种

为将甘蓝型油菜细胞质雄性不育系(Ogura CMS)的恢复基因转至甘蓝Ogura CMS材料上,以6份甘蓝Ogura CMS材料为母本,以含恢复基因的甘蓝型油菜为父本,人工杂交结合胚挽救培养,研究取材时期、培养基成分和杂交组合对胚珠成苗的影响,同时对胚挽救培养获得的植株是否为真实F1杂种进行鉴定。结果发现,胚珠培养以剥蕾授粉后16d取材时胚珠成苗数最多,成苗率为4.56%;培养基以MS+GA 0.1 mg·L^-1+NAA 0.1 mg·L^-1+0.5%水解酪蛋白(CH)+0.5%活性炭(AC)成苗效果最好,成苗率高达6.24%;M09CMS×RFO-46组合成苗数最多,成苗率为5.96%。67株胚挽救培养得到的植株经流式细胞仪、SSR分子标记和形态学鉴定,得出65株为真杂种,真杂种率高达97%。     

基于家族基因分析的甘蓝MLPK互作PUB蛋白的筛选

MLPK(M–位点受体激酶)是芸薹属自交不亲和正向调控关键元件,其参与自交不亲和信号传导的分子机制尚不明确,同时自交不亲和下游信号元件也有待于进一步分离。为了探索分离MLPK互作蛋白的思路和方法,构建了不含核定位信号的MLPK短截蛋白(MLPK-T),并利用酵母双杂交检测到MLPK与臂重复蛋白1(ARC1)作用,通过全基因组鉴定分别获得了96个甘蓝、101个白菜、70个琴叶拟南芥和62个拟南芥PUB蛋白,其中含有臂重复序列的PUB蛋白共为127个。通过系统进化分析,筛选到8个含臂重复序列的甘蓝BoPUB蛋白,其8个基因全部在柱头内表达,且成功利用酵母双杂交检测到MLPK与3个含臂重复序列的BoPUB蛋白Bol008579、Bol016165和Bol023511相互作用。     

Effects of monocrotaline-induced pulmonary hypertension on expression of Hippo signaling pathway-related molecules in rat lung

AIM:To investigate the expression of Hippo signaling pathway-related molecules in the lung tissues of the rats with pulmonary hypertension induced by monocrotaline for exploring the significance of Hippo signaling pathway in the development of pulmonary hypertension. METHODS:SD rats (n=45) were randomly divided into control group (n=15) and model group (n=30). The rats in model group was given neck subcutaneous injection of monocrota-line at 60 mg/kg to establish pulmonary hypertension model, and the rats in control group was injected with the same volume of normal saline. Four weeks later, right ventricular systolic pressure (RVSP) was measured by right cardiac catheterization, and right ventricular hypertrophy index (RVHI) and right ventricular mass index (RVMI) were calculated. The remodeling of the pulmonary arterioles was observed by HE staining, and medial thickness/external diameter (M/E%) was evaluated. The fibrosis of lung tissues was detected by Masson staining. The protein expression of Yes-associated protein (YAP), tafazzin (TAZ) and TEAD was detected by immunohistochemistry, and the protein and mRNA levels of YAP, TAZ and TEAD in lung tissues were determined by Western blot and RT-qPCR. RESULTS:Compared with control group, the vascular wall in model group was thickened significantly, the M/E% was increased (P<0.01), the pulmonary fibrosis was obvious, and the RVSP and RVHI in model group were significantly higher than those in control group (P<0.01). The immunohistochemical staining showed that the protein expression of YAP, TAZ and TEAD in the pulmonary arterioles in model group was significantly higher than that in control group. The YAP, TAZ and TEAD protein and mRNA levels in the lung tissues were also higher than those in control group (P<0.05). CONCLUSION:The activation of Hippo signaling molecules may promote the remodeling of pulmonary arterioles and further regulate the development of monocrotaline-induced pulmonary hypertension.     

Cepharanthine induces autophagy via PI3K/AKT/mTOR signaling pathway in ovarian cancer SKOV3 cells

AIM: To investigate the autophagy of human ovarian cancer SKOV3 cells induced by cepharanthine and to explore its mechanism. METHODS: The effect of cepharanthine on the viability of ovarian cancer SKOV3 cells was measured by CCK-8 assay. The SKOV3 cells were treated with cepharanthine, and then the formation of autophagosome was observed with acridine orange staining under fluorescence microscope. The protein levels of LC3, AKT, p-AKT, mTOR, p-mTOR and GAPDH in the SKOV3 cells treated with cepharanthine were determined by Western blot.RESULTS: Cepharanthine significantly inhibited the viability of ovarian cancer SKOV3 cells in a dose-dependent manner (P<0.05). The number of the intracellular acidic autophagosomes with bright red fluorescence was significantly increased after cepharanthine treatment in the SKOV3 cells. The expression of LC3-Ⅱ in SKOV3 cells was significantly enhanced after cepharanthine treatment. Furthermore, treatment with cepharanthine in the SKOV3 cells also resulted in a significant down-regulation of phosphorylated form of AKT and mTOR (P<0.01), while the total protein level was not changed. Combination of cepharanthine and 3-methyladenine resulted in a substantial decrease in the cell viability compared with using cepharanthine alone.CONCLUSION: Cepharanthine significantly inhibits the growth of human ovarian cancer SKOV3 cells and induces the autophagy, which may be correlated with down-regulation of PI3K/AKT/mTOR signaling pathway.     

NRF2 attenuates oxidative stress and lysosomal dysfunction in doxorubicin-induced H9C2 cells

AIM:To study the effect of nuclear factor E2-related factor 2 (NRF2) on oxidative stress injury and lysosomal dysfunction in doxorubicin (DOX)-induced rat myocardial H9C2 cells. METHODS:The H9C2 cells were treated with DOX. The expression of NRF2 at mRNA and protein levels was determined by real-time PCR and Western blot. The H9C2 cells stably over-expressing NRF2 were established by lentiviral infection. Real-time PCR and Western blot were used to identify the efficiency of over-expression. After DOX treatment, the cell viability was measured by CCK-8 assay, the activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and the content of malondialdehyde (MDA) in the cell supernatant were detected. FITC-dextran was used to analyze lysosomal pH, and the protein expression of lysosomal-associated membrane protein 1 (LAMP1) and cathepsin B was determined by Western blot.RESULTS:The expression of NRF2 at mRNA and protein levels in DOX-treated H9C2 cells was significantly decreased (P<0.05). Over-expression of NRF2 significantly up-regulated the mRNA and protein expression of NRF2 in DOX-treated H9C2 cells (P<0.05). After DOX treatment, the cell viability was decreased, and LDH activity was increased. The activity of SOD, GSH-Px and CAT was decreased, and the content of MDA was increased (P<0.05). The lysosomal pH was increased, and the protein expression of LAMP1 and cathepsin B decreased (P<0.05). Over-expression of NRF2 increased the cell viability, decreased LDH activity, increased the activity of SOD, GSH-Px and CAT, and decreased the content of MDA in cell supernatant (P<0.05). Over-expression of NRF2 also decreased the lysosomal pH, and increased the protein expression of LAMP1 and cathepsin B (P<0.05). CONCLUSION:DOX inhibits the expression of NRF2 in the myocardial H9C2 cells. Over-expression of NRF2 attenuates oxidative stress and lysosomal dysfunction in the H9C2 cells induced by DOX.     

Suramin alleviates hypertrophic scar in mice by inhibiting fibroplasia and inflammatory response

AIM: To investigate the therapeutical effect of suramin on hypertrophic scar (HPS) and its mechanism. METHODS: After the mouse model of HPS was established by mechanical stretching, the suramin solution at low dose (5 mg/kg) and high dose (10 mg/kg) was applied onto the scar site caused by mechanical load in mice by transdermal administration once a day for 10 d. The degree of scar hyperplasia was observed by macroscopy. The scar cross-sectional area and scar elevation index in the HPS tissues were evaluated by hematoxylin-eosin (HE) staining. The expression levels of transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6) in HPS tissues were detected by immumohistochemical staining. The expression level of α-smooth muscle actin (α-SMA) in HPS tissues was detected by immunofluorescence staining, RT-qPCR and Western blot. The levels of tumor necrosis factor-α (TNF-α), IL-6, IL-10 and TGF-β1 in the HPS tissues were measured by ELISA. RESULTS: Macroscopic observation showed that the surface areas of scar in the HPS mice after treatment with suramin at low and high doses were significantly reduced (P<0.01). HE staining results showed that the scar cross-sectional area and the scar elevation index of HPS mice after treatment with suramin at low and high doses were significantly reduced (P<0.05 or P<0.01). The results of immunofluorescence staining, RT-qPCR and Western blot showed that the number of α-SMA positive cells and the mRNA and protein expression of α-SMA in scar tissues of HPS mice after treatment with suramin at low and high doses were significantly decreased (P<0.05 or P<0.01). The results of immunohistochemical staining showed that the expression levels of TGF-β1 and IL-6 in scar tissues of HPS mice after treatment with suramin at low and high doses were significantly reduced (P<0.01). The results of ELISA showed that the levels of TNF-α, IL-6, IL-10 and TGF-β1 in the scar tissues of HPS mice after treatment with suramin at low and high doses were significantly reduced (P<0.01). CONCLUSION: Suramin inhibits the formation of HPS, which may be related to the inhibition of fibroplasia and reduction of local inflammatory response.     

马铃薯高世代选系疮痂病抗性评价及SSR辅助筛选

利用国外种质资源创造的20 份高世代马铃薯无性系为试材,以尤金为感病对照,克新18 号为抗病对照,采用自然 病圃和人工接种的鉴定方式进行疮痂病抗性评价,利用SSR 分子标记分析试材之间的亲缘关系,并对比SSR 遗传相似性系 数聚类结果和试材抗病性分类结果,探究利用SSR 分子标记辅助筛选疮痂病抗病资源的可行性。从试验地土壤和感病块茎 共分离纯化具有链霉菌特征的菌株278 份,其中240 份来自于块茎,38 份来自于土壤。具有致病力的菌株127 株,经鉴定 全部属于Streptomyces scabies 菌种。试材间的抗病性存在明显差异,可分为高抗、中抗、中感和高感4 个类型。人工接种与 自然病圃抗性鉴定结果存在极显著相关（R²=0.946 7）。抗病类型与感病类型通过SSR 遗传相似性系数聚类大致可以区分。     

新型恒温混水器换热性能数值研究

在对比分析新型混水器换热原理的基础上,利用数值模拟技术对换水器内冷热水的三维流动换热过程进行了数值研究.数值计算过程采用二阶有限体积法离散控制方程,SIMPLE求解方法以及标准k-ε模型,并对计算网格进行网格无关性验证.数值计算结果显示:新型混水器可在保证混水换热效果的同时降低混水器的能量损失,且随着流量的增加本混热结构的节能效果逐渐提升.     

论加强林业资源管理促进生态农业健康发展

生态林业建设是社会生态系统稳定发展的重要基础,需要引起人们足够的重视.现阶段我国林业资源管理中还存在着诸多的问题,制约着生态林业的发展,需要积极创新,加强林业资源管理,推动生态林业的健康发展.