三角帆蚌细胞周期蛋白基因筛选及其表达分析

细胞周期蛋白(cyclin)对细胞生长繁殖起到关键的调节作用。该研究采用高通量测序技术,构建了三角帆蚌(Hyriopsis cumingii)转录组文库,进行了cyclins基因筛选与生物信息分析,通过qRT-PCR技术,探讨其在不同组织(闭壳肌、内脏团、外套膜、斧足、心脏、血液、鳃、性腺)与性别中的表达特征。转录组测序共得到257 457条unigene(N50为1 796 bp),注释率为86.7%;通过分析,cyclin A、cyclin D2、cyclin E被筛选出,且cyclin A、cyclin D2与cyclin E蛋白的氨基酸序列均与美洲牡蛎(Crassostrea virginica)、太平洋牡蛎(Crassostrea gigas)具有较高相似性。实时荧光定量分析结果表明,cyclin A、cyclin D2、cyclin E在三角帆蚌各组织中均有表达,且存在显著的组织差异:cyclin A、cyclin E基因在性腺中表达量显著高于其他组织(P<0.05),在斧足中表达量最低;cyclin D2基因在鳃中表达量最高(P<0.05),在血液中表达量最低。在同一组织的不同性别之间:cyclin A基因在性腺、闭壳肌、心脏、血液中表达存在显著性别差异(P<0.05);cyclin D2在外套膜、心脏、性腺中的表达具有显著(P<0.05)的性别差异;cyclin E在性腺、斧足、鳃、心脏中的表达具有显著(P<0.05)的性别差异;cyclin A、cyclin E基因在雌性心脏和性腺中表达量显著(P<0.05)高于雄性,而cyclin D2反之(P<0.05),cyclin D2基因在雌性外套膜表达量显著高于雄性(P<0.05),暗示cyclin A、cyclin E与性腺发育关系密切,cyclin D2可能与鳃损伤修复有关。本研究初步探究了三角帆蚌cyclin A、cyclin D2、cyclin E基因的功能,为建立三角帆蚌细胞系提供了分子基础。     

不同黑胫病发病程度下植烟根际土壤酶活性及细菌群落结构差异比较

土传病害的发生与土壤环境,特别是微生物群落结构变化密切相关,田间自然发病条件下探讨土传病害发生、发展过程中土壤环境因子的变化特征,可更加科学地评价病害发生与土壤环境因子变化的关系,为进一步了解土传病害发生机理提供必要科学依据。选择云南烟草黑胫病危害较为严重的3个植烟区(石林板桥、保山耇街、保山珠街)统一种植高感黑胫病烤烟品种红花大金元,调查定点田块病害发生情况并根据病害调查标准将发病烟株划分不同程度(正常烟株、轻度发病烟株、中度发病烟株、重度发病烟株)。同时在保山耇街试验点采集不同发病程度烟株根际土壤,分析比较其土壤酶活性及细菌群落的差异。研究结果表明:各调查点不同发病程度烟株根际土壤与脲酶、蔗糖酶、酸性磷酸酶、过氧化氢酶活性随发病程度增加均呈下降趋势;土壤16S rRNA高通量测序及多样性分析表明,与土壤细菌门类水平优势群落组成相似,并且随发病程度放线菌门相对丰度呈下降趋势,而酸杆菌门呈上升趋势;不同发病程度根际土壤细菌群落属水平上相对丰度差异较大,其中甾体杆菌属(Steroidobacter)、阿达尔杆菌属(Adhaeribacter)、根瘤菌属(Mesorhizobium)、杜氏杆菌属(Tumebacillus)随发病程度呈先下降后上升的趋势;细菌群落α多样性指数均不同程度低于健康根际土壤,且均表现为轻度发病烟株根际土壤α多样性指数最低,其中shannon和simpson指数轻度发病烟株显著低于健康烟株;PCoA主成分分析表明,根际土壤细菌群落结构随发病程度发生了明显分离,且沿第二主成分坐标轴上细菌群落结构分离随发病程度呈逐渐增大趋势。综上所述,不同发病程度烟株根际主要环境因子存在较大差异,土壤酶活性受到不同程度抑制,细菌群落组成发生趋向性变化,群落结构发生趋向性改变,土壤微生物多样性降低。     

除草剂减量施用对麦田杂草的防治效果及对叶绿素SPAD值的影响

探讨除草剂减施对麦田杂草的防治效果,以及对小麦生长过程中叶绿素SPAD值变化的影响。结果表明,药后45 d,10%苯磺隆WP 9.00 g/hm~2处理(减施60.00%)、10%苯磺隆WP 9.00 g/hm~2+5%(唑啉草酯+炔草酯)EC 30.00 g/hm~2处理(减施60.00%)、7%双氟·炔草酯OD 63.00 g/hm~2处理(减施25.00%)和28%唑草·苯磺隆WP 16.80 g/hm~2处理(减施33.33%)对麦田阔叶杂草的鲜质量防效分别为98.64%、98.99%、98.54%和98.68%,表明除草剂减量施用对麦田杂草仍具有较好的防效。施用除草剂后,小麦叶片叶绿素SPAD值呈现先下降再上升的趋势。药后3 d、5 d、7 d,28%唑草·苯磺隆WP 33.60 g/hm~2处理(增施33.33%)小麦叶片叶绿素SPAD值显著低于7%双氟·炔草酯OD 63.00 g/hm~2处理(减施25.00%)、10%苯磺隆WP 9.00 g/hm~2处理(减施60.00%)和空白对照(CK)的叶绿素SPAD值,低于其他处理的叶绿素SPAD值。     

种植密度对2个青稞品种抗倒伏及秸秆饲用特性的影响

种植密度是影响青稞抗倒伏和秸秆饲用特性的重要因子。以抗倒伏品种昆仑14号和倒伏品种门源亮蓝为试验材料,比较研究种植密度对这2个品种生长发育、抗倒伏特性和秸秆饲用特性的影响。结果表明,种植密度对2个品种的抗倒伏和秸秆饲用特性的影响存在差异。随着种植密度的增加,昆仑14号根长、根体积、根数和根干重先增后降,茎粗和壁厚依次下降;门源亮蓝根系和茎秆相关指标则随种植密度增大而下降。昆仑14号抗倒伏相关指标先增后降,但整个生育期未发生倒伏;门源亮蓝各指标均显著降低,诱发倒伏现象提前发生,致使倒伏率增大、倒伏程度加剧。昆仑14号茎秆中性洗涤纤维、酸性洗涤纤维、半纤维素、纤维素和木质素等化学成分含量随密度增加先增后降,门源亮蓝各成分含量呈下降趋势,相对饲喂价值随密度增加呈现增高的趋势。综合抗倒伏特性与秸秆饲用特性,昆仑14号最佳种植密度为375×104株hm~(-2),门源亮蓝粮饲兼用时适宜密度为300×104～375×104株hm~(-2)。     

孕穗期低温灌溉对水稻剑叶和颖花生理特征的影响

为探究孕穗期持续低温对水稻的伤害机理,利用Y两优957、天优华占、Y两优1号和中早39这4个生产上大面积推广的水稻品种,分别在其幼穗分化Ⅴ期进行17.5℃、10 d的低温冷水灌溉处理。处理结束当天利用SPAD502测定剑叶SPAD值、利用酶联免疫吸附试验测定剑叶与颖花的抗氧化系统和渗透调节系统等生理生化指标、液相色谱串联质谱法测定ABA含量。结果表明,孕穗期低温胁迫降低了水稻的结实率与花粉育性,但不同品种的降幅有差异,分析认为结实率下降的重要原因之一是花粉育性下降。孕穗期低温胁迫后剑叶SPAD值降低,且不同品种表现有差异。根据结实率的结果,Y两优957和天优华占可作为低温耐受品种,Y两优1号和中早39可作为低温敏感品种。低温处理后,低温耐受品种剑叶SOD、POD、CAT活性升高,颖花SOD活性降低;低温敏感品种剑叶SOD、POD活性降低,颖花SOD活性升高,与低温耐受品种相反。低温耐受品种低温处理后渗透调节物质在剑叶中大多高于对照,颖花中则均低于对照;低温敏感品种则呈现较为相反的趋势。低温处理后无论材料是否耐受低温,其颖花中ABA含量均显著高于对照组。颖花与剑叶在抗氧化系统的SOD活性、渗透调节系统的PRO和STS含量等多个生理指标上大多呈现较为相反的变化趋势,推测水稻的源库受低温胁迫后可能存在一个动态平衡调节机制,为研究孕穗期低温胁迫对水稻生理特征影响提供了新的思路。     

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.     

Autophagy induces mitochondrial dysfunction in neurons from neonatal rats with hypoxic ischemic encephalopathy

AIM: To explore the influence of autophagy on the induction of mitochondrial dysfunction in the neurons in a neonatal rat hypoxic ischemic encephalopathy (HIE) model. METHODS: Ten-day-old rat pups (n=30) were randomly divided into sham group and model group. The rats in the latter group were subject to hypoxia-ischemia treatment via unilateral common carotid artery ligation. The rats were sacrificed for brain pathological examination, and the protein levels of cleaved caspase-3 and LC3B-II were detected by immunohistochemical analysis. For the in vitro experiments, the autophagy of primarily cultured rat neurons was observed after hypoxia, and Western blot and mitochondrial function testing were also performed. RESULTS: Compare with sham group, the hypoxia-ischemia treatment caused atrophy and apoptosis of neurons, and ventricular area enlargement of rat brains. Immunohistochemical results demonstrated significantly higher levels of apoptosis- and autophagy-associated proteins, such as cleaved caspase-3 and LC3B-II (P<0.01). In vitro experiments demonstrated that hypoxia induced autophagy and apoptosis in the neurons. Compared with sham group, there were higher levels of reactive oxygen species and mitochondrial superoxide, and lower mitochondrial membrane potential in the model group (P<0.01). CONCLUSION: In neonatal HIE rat model, the hypoxia-induced mitochondrial dysfunction is related to apoptosis and autophagy. It will provide a new idea for administration of autopahgy inducer agents in treatment of HIE.     

vvi-miR160s介导VvARF18应答赤霉素调控葡萄种子的发育

【目的】研究vvi-miR160家族（vvi-miR160s）及其靶基因在‘魏可’葡萄种子发育过程中的作用,探究其应答赤霉素（GA）调控葡萄果实无核的潜在机理。【方法】采用miR-RACE、生物信息学分析、RT-qPCR、RLM-RACE等方法,鉴定vvi-miR160家族成员及其靶基因,分析vvi-miR160s及其靶基因应答GA的时空表达模式及其潜在功能。【结果】花前GA处理强烈抑制‘魏可’葡萄胚珠及种子发育,高效诱导其无核,且无核率达99.8%。克隆鉴定了VvMIR160s前体基因序列（501 bp）及成熟体序列,且它们在不同物种间具有较高的保守性;基于vvi-miR160s序列,预测到靶基因VvARF18,利用RLM-RACE技术检测到vvi-miR160s对VvARF18的裂解位点在第10和第11位之间,裂解频度9/17,证明VvARF18是vvi-miR160s的真实靶基因。该靶基因编码683个氨基酸,在398—411位存在核定位信号,且该蛋白亚细胞定位于细胞核上。VvARF18与其他物种间序列的同源保守性较高,基因结构相似,其中VvARF18蛋白与茶、烟草、梅花等物种亲缘关系较近。VvARF18启动子中包含4种作用元件,且含有较多激素相关元件。RT-qPCR结果显示,随着葡萄果实的发育,vvi-miR160c/d/e呈现‘V’形表达趋势,在硬核种子发育期表达较低,而VvARF18表现出与前者相反的表达趋势,在硬核种子发育期呈现高表达,表明vvi-miR160c/d/e对VvARF18负调控,但vvi-miR160a/b与VvARF18表达水平却不存在明显负相关。GA处理极显著地上调了vvi-miR160a/b在葡萄硬核种子发育期的表达,同时也显著抑制了VvARF18在同时期的表达,且它们的表达水平呈负相关,表明GA处理促进了vvi-miR160a/b对VvARF18的负调控作用;相反,GA减弱了vvi-miR160c/d/e对VvARF18的负调控作用。【结论】在vvi-miR160家族中,vvi-miR160c/d/e可能介导VvARF18在葡萄种子发育的特定阶段调控种子的发育形成,而vvi-miR160a/b可能主要介导VvARF18参与调控GA诱导葡萄种子败育的过程。     

饲用燕麦和草木樨混播栽培技术

为了提高单位面积土地的利用效率，增加单位面积经济效益，采用饲用燕麦和草木樨混播技术，不仅可以合理、有效地利用空间、光照、热量和水分等资源，增加牧草产量，提高牧草品质，还可以进行营养互补；草木樨还可以改善土壤结构、提高土壤肥力。通过开展饲用燕麦和草木樨混播栽培技术等方面的综合研究，明确了饲用燕麦和草木樨混播栽培选地、种子准备、播种、田间管理、收获等关键技术指标，为大田生产的规范化、标准化、产业化、市场化提供了技术支撑。     

盐酸多西环素片在羔羊体内残留消除规律及休药期分析

旨在确定盐酸多西环素片按照给药说明给药后在羔羊体内的残留消除规律及休药期。将盐酸多西环素片根据体重以5 mg·kg^-1内服给药，间隔24 h，连续给药5次。在最后1次给药后，分别在第0（12小时）、1、2、3、5、7和9天时间点采集羔羊脂肪、肌肉、肝和肾，采用建立并验证的HPLC-VWD方法测定组织中多西环素的含量。结果显示：方法学考察结果表明，在50~5 000 ng·mL^-1添加的线性方程和相关系数为y=0.044x-0.414，R²=0.999。试验结果表明，多西环素在羔羊组织中代谢快速，最后1次给药后第9天，在肌肉、肝、肾和脂肪中均未检测到多西环素。本试验以5 mg·kg^-1体重内服给予羔羊盐酸多西环素片后，根据欧洲药品评估机构法规《EMEA/CVMP/036/95》，建议盐酸多西环素片在羔羊组织中的休药期为2 d。