利用CRISPR/Cas9敲除葡萄VviPDS1基因的研究

选择葡萄八氢番茄红素脱氢酶(Phytoene desaturase)基因VviPDS1为靶标,利用CRISPR/Cas9系统构建基因敲除载体,瞬时转化葡萄叶片原生质体,检测到不同类型的突变。通过农杆菌介导转化‘无核白’葡萄胚性愈伤组织,筛选获得卡那霉素抗性植株71株。经PCR鉴定,其中53株为阳性植株,阳性率为74.64%。测序结果表明,共有20株在靶点发生不同类型的突变,编辑效率为37.74%;其中9株产生了双等位基因突变。对其进行氨基酸序列预测,在第202位氨基酸之后发生了不同程度的变异。利用CRISPR/Cas9系统敲除VviPDS1获得的突变体植株呈现整体矮化,其叶片出现不同程度白化。表明CRISPR/Cas9系统可以通过细胞中的瞬时或稳定表达进行基因编辑,可以实现在葡萄编辑植株中产生纯合敲除。     

Astrocyte activity and autophagy after radiation-induced brain injury in rats

AIM: To investigate the activity of astrocytes and autophagy-related changes after radiation-induced brain injury (RBI) in rats.METHODS: A total of 36 Sprague-Dawley rats, weighing 180~200 g, were trained for 4 d in the Morris water maze. They were randomly divided into sham group, model group and 3-methyladenine (3-MA) group. The rats in model group and 3-MA group were given single whole-brain X-ray irradiation at a dose of 20 Gy after intraperitoneal anesthesia. After the irradiation was completed, the rats in model group was given 5 μL of NaCl into the lateral ventricle, and the rats in 3-MA group was injected with 3-MA at 600 nmol into the lateral ventricle. After 8 weeks of feeding, Morris water maze was used for measuring the learning and memory abilities. The brain tissues were taken and HE staining was used to observe the pathological changes of the hippocampus. The protein level of GFAP was determined by immunohistochemistry and Western blot for evaluating astrocyte activity. Dual fluorescence staining of GFAP and LC3 was performed for evaluating the changes of autophagy in the astrocytes. The protein level of cleaved caspase-3 detected by Western blot and TUNEL staining in the ipsilateral hippocampus were used to evaluate the apoptosis. The contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were examined by ELISA to assess the inflammatory response in the hippocampus.RESULTS: Radiation inhibited astrocyte activity, activated autophagy in astrocytes, and aggravated brain damage. 3-MA promoted the activation of astrocytes and promoted the repair of brain tissue damage.CONCLUSION: The injury of rat hippocampus after radiation is obvious, and the number of astrocytes is significantly reduced. 3-MA significantly attenuates the damage. This finding may provide a new approach for the treatment of radiation-induced brain injury.     

甜樱桃花芽不同发育时期内参基因的筛选与验证

为了筛选甜樱桃花芽不同发育时期均稳定表达的内参基因,以甜樱桃桑提娜和黔樱一号不同发育时期花芽为材料,通过qRT-PCR技术检测28S rRNA、EF1-a1、EF1-a2、UBC、RPL13、18S rRNA、RSP3、CYP40、ACT2和α-TUB3等10个常用看家基因的表达水平,并利用GeNorm、NormFinder和BestKeeper综合评价其表达稳定性。结果表明,EF-1a2和RSP3在所有样品中稳定性最好。分别以EF-1a2、RSP3及EF-1a2+RSP3作为内参基因检测不同发育时期花芽生长素运输载体AUX1基因及生长素响应因子ARF基因的表达模式,该2个基因在不同内参基因标定下表达模式相同。表明EF-1a2、RSP3及EF-1a2+RSP3可作为甜樱桃花芽不同发育时期的内参基因。     

大功率气体发动机电子节气门设计与研究

以大功率气体发动机的电子节气门为研究对象,对其进行了系统的设计和研究。在分析电子节气门机械结构的基础上,建立了电子节气门数学模型,采用冗余设计思想设计了双H桥电子节气门驱动电路。采用增量式PID控制算法对电子节气门开度进行精确的闭环控制。实验结果表明,设计的电子节气门系统鲁棒性好,具有很好的响应性和稳定性,能满足大功率气体发动机的性能需求。     

Potassium accumulation,partitioning, and remobilization in high-yield spring maize in Northeast China

Potassium (K) plays an important role in maize yield, but K accumulation characteristics in high yielding maize is still not well documented. A two-year field experiment was conducted to investigate the K accumulation characteristics of high yield (HY)（>15 t ha^?1）spring maize compared with medium yield (MY)（10–15 t ha^?1), and low yield (LY)（<10 t ha^?1). The maximum K accumulation stage in maize appeared between V6 and V12 stage (LY, MY, and HY was 125.46, 138.05, and 146.22?kg ha^?1, respectively). Most of the K (94.27–97.13%) was accumulated during vegetative stages. HY exhibited a significantly higher K accumulation than either MY or LY. For different organs, the K remobilization amount of leaf was the highest, and the remobilization amount of stalk was the lowest, the K remobilization amount of leaf and stalk showed as HY?<?MY?<?LY, same as the total K remobilization amount, but the K remobilization amount of sheath and husk plus cob showed the opposite order. These results indicated that sufficient nutrient supply for maize can not only accumulate more K but also delay leaf senescence and maintain high photosynthetic activity, resulting in reduced K remobilization from vegetative organs, and reduced K loss in whole plant.     

不同栽培密度和钾钠离子处理对基质培番茄产量与品质的影响

以优质番茄品种京采6 号为试材，研究了不同栽培密度（3.8、5.0 株 · m^-2）与离子处理（K⁺、Na⁺）对基质培番茄生长、产量与品质的影响，构建了番茄果实品质综合评价指数TQI。结果表明：提高营养液中的K⁺ 浓度，能够在不影响产量的同时增加番茄第2 穗果实可溶性糖、可溶性固形物、糖酸比和VC 含量；栽培密度对番茄产量和品质的影响较小；栽培密度× 离子互作显著影响了第1 穗果实有机酸、亚硝酸盐含量与糖酸比；第2 穗果是生产高品质番茄的关键，其在3.8 株 · m^-2与高K+ 营养液条件下可获得最高的TQI。综合来看，建议在实际生产中控制栽培密度为3.8 株 · m^-2，同时采用高K+ 营养液灌溉，可在稳产条件下获得高品质番茄。     

Cf-2/Rcr3pim番茄品系对南方根结线虫的抗性测定

Cf-2/Rcr3~(pim)基因型番茄不仅能够抵御番茄叶霉菌的侵染,而且对马铃薯金线虫的寄生也有一定的抑制效果。为挖掘根结线虫的新抗性资源,本研究采用室内人工接种法测定了Cf-0/Rcr3~(pim)、Cf-2/Rcr3-3和Cf-2/Rcr3~(pim)基因型番茄品系对南方根结线虫的抗感性。抗性评价结果显示,Cf-0/Rcr3~(pim)品系对南方根结线虫表现高感,Cf-2/Rcr3-3品系为中感,而Cf-2/Rcr3~(pim)品系则为感病。与Cf-0/Rcr3~(pim)和Cf-2/Rcr3-3基因型相比,Cf-2/Rcr3~(pim)基因型番茄品系虽然对南方根结线虫侵染的敏感性略低,但是不能阻止线虫在根系上的大量繁殖,不适于根结线虫的防控应用。     

Effect of Linc00152 targeting miR-376c-3p on viability,apoptosis and radiosensitivity of cervical cancer cells

AIM: To investigate the effect of Linc00152 on the viability, apoptosis and radiosensitivity of cervical cancer cells. METHODS: RT-qPCR was used to detect the expression levels of Linc00152 and microRNA-376c-3p(miR-376c-3p) in human cervical cancer HeLa cells and SiHa cells, and normal cervical Ect1/E6E7 cells. The cervical cancer HeLa cells with low Linc00152 expression or miR-376c-3p over-expression were established. MTT assay, flow cytometry, colony formation assay and Western blot were used to determine the cell viability, apoptosis, radiosensitivity and related protein expression. The dual-luciferase reporter assay was used to verify the regulatory relationship between Linc00152 and miR-376c-3p in the HeLa cells. RESULTS: Compared with the Ect1/E6E7 cells, Linc00152 was up-regulated in the HeLa cells and SiHa cells, and miR-376c-3p was down-regulated (P < 0.05). Low expression of Linc00152 or over-expression of miR-376c-3p inhibited the viability of HeLa cells, induced apoptosis, enhanced the radiosensitivity, inhibited the protein expression of cyclin D and Bcl-2, and promoted the protein expression of P21 and Bax (P < 0.05). Linc00152 negatively regulated miR-376c-3p expression in the HeLa cells, and inhibition of miR-376c-3p expression reversed the effect of low expression of Linc00152 on HeLa cell viability, apoptosis and radiosensitivity. CONCLUSION: Linc00152 is highly expressed in the cervical cancer cells. Linc00152 affects the viability, apoptosis and radiosensitivity of HeLa cells by targeting miR-376c-3p, which is a potential diagnosis and treatment target for cervical cancer.     

Sulodexide protects against hyperglycemia-induced retinal vascular injury via suppressing NOX4/NLRP3 pathway

AIM To investigate the effect of sulodexide (SDX) on high glucose-induced damage in retinal microvascular endothelial cells. METHODS (1) High-fat diet combined with intraperitoneal injection of streptozocin were used to induce type 2 diabetes mellitus (DM) followed by injection of saline or SDX in C57BL/6J male mice. Retinal microvascular leakage and density, and the protein levels of NLRP3 inflammasome-related proteins, zonula occludens-1 (ZO-1) and NADPH oxidase 4 (NOX4) were measured. (2) Human retinal microvascular endothelial cells (HRMECs) were treated with normal glucose or high glucose with or without SDX, and were further transfected with siRNA to knock down NOX4, or infected by adenovirus to over-express NOX4. The protein levels of ZO-1, VE-cadherin (VE-Cad), NOX4 and NLRP3 inflammasome-related proteins as well as the level of reactive oxygen species (ROS) were detected. RESULTS Treatment with SDX increased the protein level of ZO-1, attenuated retinal leakage and NLRP3 inflammasome activation, and enhanced the density of microvasculature and the number of ganglion cells in diabetic retinas. The protein levels of ZO-1 and VE-Cad were decreased, while the levels of NOX4, NLRP3 inflammasome-related proteins and ROS generation were increased in high glucose-treated HRMECs. Silencing of NOX4 inhibited high glucose-induced increases in NLRP3 inflammasome and ROS generation, and decreases in the protein levels of ZO-1 and VE-Cad. Over-expression of NOX4 significantly increased the levels of NLRP3 inflammasome-related proteins and ROS generation in HRMECs, and reduced the protein levels of ZO-1 and VE-Cad. Treatment with SDX partly reversed NOX4 over-expression-induced changes. CONCLUSION SDX alleviates hyperglycemia-induced retinal microvascular endothelial injury via inhibiting NOX4/ROS/NLRP3 pathways.     

Mouse melanoma cell exosomes promote expression of Rac1 protein in fibroblasts

AIM To investigate the effect of exosomes secreted by mouse melanoma cells on the expression of Ras-related C3 botulinum toxin substrate 1 (Rac1) protein in fibroblasts. METHODS Ultracentrifugation was adopted to separete exosomes secreted by mouse melanoma B16-F10 cells. The morphological structure of exosomes was observed by negative-staining electron microscopy. The size distribution of exosomes was determined by nanoparticle tracking analysis (NTA). The exosomal markers, tumor susceptibility gene 101 (Tsg101) and tyrosinase-related protein 2 (Tyrp2), were identified by Western blot. Laser confocal microscopy was used to observe the process that mouse embryonic fibroblasts (MEF) took in exosomes during co-culture. Immunocytochemical staining and Western blot were used to detect the expression of Rac1 protein in MEF. RESULTS B16-F10 cell exosomes showed a typical tea tray-like structure, with a size range of 141~255 nm, and expressed protein markers Tsg101 and Tyrp2. The results of laser confocal microscopy showed that compared with co-culture at 0 h, a small number of exosomes appeared in the MEF at 12 h, and a large number of exosomes accumulated in the MEF after co-cultured for 24 and 36 h. Western blot analysis showed that compared with co-culture at 0 h, the expression of Rac1 protein in the MEF was significantly increased at 24 h and 36 h of co-culture (P<0.01). The results of immunocytochemical staining showed that compared with co-culture at 0 h, the positive expression level of Rac1 in the MEF cells was significantly increased at 12 h, 24 h and 36 h of co-culture (P<0.05 or P<0.01). CONCLUSION Intake of exosomes secreted by mouse melanoma cells promotes the expression of Rac1 protein in fibroblasts.