番茄匍柄霉叶斑病拮抗细菌的筛选与鉴定

应用平板对峙法从连作多年的番茄根际土样中筛选得到对番茄匍柄霉叶斑病具有较强拮抗活性的细菌菌株ZF161，该菌株对番茄匍柄霉的平板抑制率达到70.51%。离体叶片试验显示，菌株ZF161对番茄匍柄霉叶斑病防治效果达到69.83%。通过菌落形态观察、生理生化特性、Biolog测定和多基因系统发育树综合分析，鉴定菌株ZF161为枯草芽孢杆菌（Bacillus subtilis）。番茄盆栽试验结果表明，菌株ZF161对番茄匍柄霉叶斑病的防治效果达到63.27%。进一步平板对峙抑菌谱试验结果显示，菌株ZF161对其他7种病原真菌也具有较好的抑制效果。上述结果说明，菌株ZF161对番茄匍柄霉叶斑病具有良好的防治效果，且对多种病原真菌也具有抑制作用，具有开发应用的潜力。     

ROCK promotes high glucose-induced cardiomyocyte apoptosis by inhi-biting PI3K/Akt signaling pathway

AIMTo investigate whether Rho-associated coiled-coil kinase (ROCK) is involved in high glucose-induced apoptosis of primary cardiomyocytes by regulating PI3K/Akt signaling pathway. METHODSPrimary Wistar rat cardiomyocytes were cultured and identified by α-sarcomeric actin (α-SCA) immunohistochemistry. Cardiomyocytes were treated with 5.5, 33 and 40 mmol/L glucose for 48 h. The cell viability was measured by MTT assay, and the mRNA expression of ROCK1 and ROCK2 in the cardiomyocytes was detected by RT-qPCR. Flow cytometry was used to analyze the apoptosis of the cardiomyocytes. The protein levels of ROCK1, ROCK2, cleaved caspase-3, Bcl-2, PI3K, Akt and p-Akt were determined by Western blot. In order to confirm the regulatory effect of ROCKs on PI3K/Akt signaling pathway, the cells were divided into control group (5.5 mmol/L glucose), high glucose group (33 mmol/L glucose) and high glucose+Y27632 (ROCK inhibitor) group. Western blot was used to detect the protein levels of ROCK1, ROCK2, PI3K, Akt and p-Akt. RESULTSAfter 48 h of high glucose exposure, the values of relative cell viability in 33 and 40 mmol/L glucose groups were (79.71±2.43)% and (68.41±7.49)%, respectively, both of which were significantly decreased compared with normal control group (P<0.05). After 48 h of high glucose exposure, the relative mRNA levels of ROCK1 and ROCK2 in 33 and 40 mmol/L glucose groups were significantly increased compared with normal control group (P<0.05). Compared with normal control group, the apoptotic rate in 33 and 40 mmol/L glucose groups was increased significantly (P<0.05). Compared with normal control group, the protein expression of ROCK1, ROCK2 and cleaved caspase-3 in 33 and 40 mmol/L glucose groups was increased (P<0.05), while the protein expression of Bcl-2 was decreased (P<0.05). No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed, while the protein level of p-Akt in 33 and 40 mmol/L glucose groups was decreased compared with normal control group (P<0.05). Compared with high glucose group, the expression of ROCK1 and ROCK2 was decreased in high glucose+Y27632 group. No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed. Compared with normal control group, the protein level of p-Akt in high glucose group was decreased, and the protein level of p-Akt in high glucose+Y27632 group was increased significantly compared with high glucose group. CONCLUSION Under high glucose environment, ROCK may reduce the level of p-Akt by inhibiting the PI3K/Akt signaling pathway， thus promoting the apoptosis of cardiomyocytes.     

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。     

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。     

FAM3C activates Akt to promote viability of oral squamous-cell carcinoma cells

AIM: To investigate the expression and roles of family with sequence similarity 3, member C (FAM3C) in oral squamous-cell carcinoma cells. METHODS: The mRNA and protein expression levels of FAM3C in dysplastic oral keratinocyte (DOK) and oral squamous-cell carcinoma WSU-HN6 cells were detected by RT-qPCR and Western blot. The WSU-HN6 cells were treated with siFAM3C or FAM3C antibody. After 24, 48 and 72 h, the viability of WSU-HN6 cells was measured by CCK-8 assay, and the activation of protein kinase B (Akt) was detected by Western blot. Adenovirus was used to mediate over-expression of FAM3C in the DOK cells. The DOK cell viability was measured by CCK-8 assay after adenovirus infection for 24, 48 and 72 h, and the activation of Akt was detected by Western blot. RESULTS: Compared with the DOK cells, the mRNA and protein levels of FAM3C were significantly increased in the WSU-HN6 cells (P<0.05). The viability of WSU-HN6 cells transfected with siFAM3C was significantly inhibited at 48 h and 72 h (P<0.05). siFAM3C treatment inhibited the activation of Akt (P<0.05). FAM3C antibody treatment also suppressed the viability of the WSU-HN6 cells at 48 h and 72 h and the activation of Akt (P<0.05). Over-expression of FAM3C in the DOK cells promoted the cell viability at 48 h and 72 h and activated Akt (P<0.05). CONCLUSION: FAM3C might promote oral squamous-cell carcinoma cell growth by activating Akt.     

山葡萄VaCIPK18原核表达与多克隆抗体制备

类钙调磷酸酶B亚基蛋白(CBLs)互作蛋白(CIPKs) 作为类丝氨酸/苏氨酸蛋白激酶,在植物响应非生物胁迫信号转导中起着重要作用。基于前期山葡萄响应低温胁迫转录组测序结果,发现低温胁迫引发的早期伤害及感知阶段的激酶基因中涉及CBL-CIPK 信号通路的VaCIPK18表达显著上调。为进一步研究山葡萄(Vitis amurensis)VaCIPK18激酶参与低温胁迫的功能,采用同源克隆获得了VaCIPK18基因,其开放阅读框为1 320 bp,编码439个氨基酸。基于对VaCIPK18蛋白生物信息学分析,获取胞外结构域中抗原表位丰富的肽段,并将其C端调控结构域(230~439 aa)构建到原核表达载体pET28a-SUMO。将重组表达载体转化至大肠杆菌(E. coli Rosetta)中,经0.8 mmol·L^-1 IPTG、37℃诱导4 h表达出大小为42 kDa的包涵体蛋白。将重组蛋白作为抗原免疫日本大耳白兔,获得anti-VaCIPK18多克隆抗体,经检测具有高效价及特异性。Western Blot结果表明,该抗体可以与葡萄内源CIPK18特异性结合,且在50 kDa位置出现与预期一致的条带。同时,CIPK18在低温胁迫后葡萄叶片中蛋白表达水平与室温下相一致,但两种状态下均存在可能的磷酸化与泛素化修饰现象。本研究结果为进一步探究VaCIPK18的蛋白定位、表达及其功能奠定了基础。     

Role of cytokeratin 8 on changes of intestinal epithelial permeability induced by corticotropin-releasing factor

AIM: To investigate the role of cytokeratin 8 (CK8) on the change of intercellular permeability of intestinal epithelial cells induced by corticotropin-releasing factor (CRF). METHODS: The expression levels of CRF receptor 1 (CRFR1) and CRFR2 on human colon adenocarcinoma HT29 cell surface were determined by immunofluorescence staining. After treatment with 100 nmol/L CRF for 72 h, the translocation of FITC-labelled dextran was measured in a Transwell chamber. The structural changes of tight junctions were observed under transmission electron microscope. The expression levels of CK8, and tight junction proteins ZO-1 and occludin were determined by Western blot. The activity of protein kinase C (PKC) was detected by ELISA. Furthermore, the effects of CRF on intestinal epithelial permeability were examined in CK8-silencing HT29 cells, which were constructed by infection with sh-CK8 lentivirus. RESULTS: CRF treatment increased the permeability of FITC-labelled dextran (P<0.05), caused the opening of tight junctions, and induced increased fluorescence intensity of CK8. The expression levels of occludin and ZO-1 were down-regulated (P<0.05). PKC activity was decreased at 1 h after CRF treatment (P<0.05). CRF-induced increase in the permeability and down-regulation of occludin were not blocked by CK8 silencing. Nevertheless,CK8 silencing blocked the effects of CRF regarding the decrease in the expression levels of ZO-1 and the increase in PKC activity (P<0.05). CONCLUSION: CK8 may be involved in CRF-induced increase in intestinal epithelial permeability by inhibiting the activity of PKC, and there may be other signaling pathways involved.     

朱顶红无菌苗叶片高效再生体系

以朱顶红（Hippeastrum vittatum）叶片为外植体进行离体培养，具有取材方便、试材充足、成本低等优势，但叶片诱导再生率极低，是朱顶红离体培养的一大难题。本试验中分别以‘花孔雀’和‘黑天鹅’朱顶红无菌苗叶片为外植体，探究了不同植物生长调节剂和不同取材部位对不定芽诱导和继代增殖的影响。结果表明：最佳外植体为 MS 培养基中培养 10 d 形成的幼嫩叶片基部（0.5 cm），在光照 16 h · d-1（光照强度 36 μmol · m-2 · s-1）下，不定芽诱导的最适培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 NAA + 2 mg · L-1 TDZ，两个品种的不定芽均以间接途径发生，其中‘花孔雀’在培养 40 d 后形成愈伤组织，55 d形成不定芽，诱导率可达 69.44%；‘黑天鹅’在培养 45 d 后形成愈伤组织，65 d 形成不定芽，诱导率达到 66.67%；最适体细胞胚诱导培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 PIC，‘花孔雀’和‘黑天鹅’的诱导率分别达到 66.67%和 63.89%；最佳不定芽增殖培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 NAA + 1 mg · L-1 TDZ，‘花孔雀’和‘黑天鹅’的增殖系数分别达到 4.67 和 3.46；在不添加植物生长调节剂的 MS培养基中进行生根培养，30 d 后两个品种的生根率均达到 100%；将生根培养 30 d 的小植株转移至室温条件下放置 3 d，摘去封口膜再驯化 3 d 后，移栽至经高温消毒的草炭︰蛭石（体积比）为 1︰1 的基质中，成活率达到 100%     

Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.