Chilling requirements of Paeonia cultivars

Dormant second year potted plants of Paeonia ‘Coral Sunset’, ‘Monsieur Jules Elie’, and ‘Sarah Bernhardt’ were placed into three chilling regimes (constant 1, 4, or 7°C) for different durations (3, 6, 9, or 12 weeks) to ascertain their chilling requirements for shoot and flower production. Chilling was followed by forcing for up to 5 weeks at 18°C, then plants were maintained in a controlled greenhouse until flowering had finished. Mean number of shoots and flowers per plant were recorded and the time taken for shoots to sprout was calculated.Control plants (forced immediately without chilling) produced no shoots or flowers. For all cultivars, the proportion of plants that sprouted, and the mean number of shoots and flowers increased as plants were subjected to colder chilling temperatures, or longer chilling durations. However, there were no significant within-cultivar differences between different treatments of 9 weeks or more. The time taken for sprouting to occur after the completion of each chilling treatment consistently decreased as the duration of the chilling treatment increased. In most cases, lower chilling temperatures lead to more rapid sprouting once plants were placed in the 18°C forcing conditions.When a simple model was fitted where the chilling temperature and duration of each treatment was described by a cumulative normal curve rising from zero to some maximum value (or potential) once adequate chilling had been received, we found that temperatures of 4 and 7°C provided only 83 and 59%, respectively, of the chilling accumulated per unit time at 1°C. ‘Coral Sunset’, an interspecific hybrid early flowering type, required the greatest amount of chilling to sprout consistently, while ‘Sarah Bernhardt’, a very late flowering type, required the least. Of the three cultivars, ‘Sarah Bernhardt’ also required the least amount of chilling to achieve its potential shoot and flower numbers, while ‘Monsieur Jules Elie’, a mid-season flowering type, required the most chilling to achieve the same end for these two variables. This suggests that the response to spring temperatures as well as chilling influences the time of flowering.     

Screening of cowpea cultivars for resistance to leaf smut

Many of the cowpea cultivars which were resistant to leaf smut disease in Brazil were found to be susceptible in Nigeria under high inoculum pressure. Out of 40 cultivars tested, three were found to be immune at Ibadan for two seasons and near Kano for one season in 1995. They were IT85F-2805, IT88S-584-1 and TVu 1235. Six cultivars—IAR 48, IT81D-1228-14, IT83D-442, IT86D-1056, TVu 4031 and TVu 11067 showed resistant reactions at Kano and at Ibadan. The above nine cultivars have high yielding characteristics, therefore they could be adopted for cultivation by farmers where leaf smut severity is high.     

玉米HD-ZIP I亚家族基因鉴定及表达分析

转录因子是植物响应逆境胁迫的重要调节因子,在其整个生长发育过程中发挥着重要的作用。HD-ZIP家族蛋白是植物中特有的一大类转录因子,包含4个亚家族(HD-ZIP I^IV),其中HD-ZIP I亚家族成员主要参与干旱、渗透压等极端环境和ABA及乙烯等激素处理的响应过程。本文采用隐马可夫模型(HMM)在玉米参考基因组中鉴定到17个HD-ZIP I亚家族成员,这些基因不均匀分布于玉米6条染色体上,与水稻的亲缘关系要近于拟南芥。玉米HDZIP I亚家族基因在玉米7种组织中表现出多种表达模式,具有明显的组织表达特异性。另外, HD-ZIP I亚家族基因对高盐、淹水及冷害等不同的逆境胁迫处理呈现出不同的响应模式及响应程度差异。5种不同激素处理后,玉米HD-ZIP I亚家族基因也表现出复杂的响应模式。这些结果为进一步解析玉米HD-ZIP I亚家族基因的生物学功能和作用机理提供了一定的参考价值。     

Pyroptosis mediates high glucose-induced inflammation and injury in MC3T3-E1 osteoblasts

AIM To investigate whether pyroptosis contributes to the inflammation and injury in mouse embryonic osteoblastic cell line MC3T3-E1 induced by high glucose (HG; 45 mmol/L glucose). METHODS The cell viability was measured by CCK-8 assay. The protein expression levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and caspase-1 (CASP1) were determined by Western blot. The secretion levels of interleukin-18 (IL-18) and IL-1β were measured by ELISA. The intracellular level of reactive oxygen species (ROS) was detected by 2',7'-dichlorodihydrofluorescein diacetate staining followed by photofluorography. Mitochondrial membrane potential (MMP) was examined by rhodamine 123 staining followed by photofluorography. The alkaline phosphatase (ALP) activity was determined using the ALP kit, and the number of mineralized nodules was detected by alizarin red S staining. RESULTS After the MC3T3-E1 osteoblasts were treated with HG for 24 h, the protein expression levels of NLRP3 and CASP1, and the secretion levels of IL-18 and IL-1β were significantly increased. The decrease in cell viability, and the increases in ROS generation and MMP loss were also observed. Moreover, the differentiation and mineralization of MC3T3-E1 osteoblasts were inhibited, evidenced by decreases in both ALP activity and mineralized nodule number. Knockdown of CASP1 by siRNA attenuated the HG-induced osteoblast inflammation and injury mentioned above. CONCLUSION Pyroptosis mediates HG-induced inflammation and injury in MC3T3-E1 osteoblasts.     

SRT1720 enhances maturity and quality of oocytes in aged mice

This study aims to investigate the morphology and distribution of mitochondria, spindles, and chromosomes in oocytes of aged mice and examine the effects of SRT1720 on oocyte maturation. C57BL/6J mice were divided into young (4–8 weeks) and aged groups (48–52 weeks). In vitro maturation media contained (0.05, 0.1, and 1.0 μM) SRT1720 and 0.1-μM dimethyl sulfoxide (DMSO control). The rate of chromosome misalignment and spindle misorientation in oocytes of aged mice were significantly higher than that of young mice (P < 0.01). Fluorescence intensity of mitochondria from oocytes of aged mice was significantly lower than that of young mice (P < 0.01). SRT1720 at 0.1 μM significantly improved oocyte maturation, fertilization, and blastocyst formation in aged mice compared with young mice (P < 0.01). Additionally, immunofluorescence intensity of mitochondria, normal spindle morphology, and chromosome alignment were notably enhanced with SRT1720 when compared with the DSMO control group for metaphase II (MII)-stage oocytes matured in vitro (P < 0.01); 0.1-μM SRT1720 enhanced the expression level of SRIT1 in oocytes from aged mice. In summary, the aged mice oocytes showed increased nuclear and cytoplasmic defects, whereas SRT1720 enhanced oocyte maturation and quality. We concluded that 0.1-μM SRT1720 was an appropriate concentration for in vitro maturation media.     

Sublethal effect of avermectin and acetamiprid on the mortality of different life stages of Brontispa longissima (Gestro) (Coleoptera: Hispidae) and its larvae parasitoid Asecodes hispinarum Bouček (Hymenoptera: Eulophidae)

The beneficial parasitoid Asecodes hispinarum Bouček plays an important role in integrated pest management (IPM) of the coconut leaf beetle, Brontispa longissima (Gestro), in China. A. hispinarum females parasitize 3rd to 4th instars B. longissima larvae. Hatched parasitoid larvae develop within the host, and parasitoid adults emerge through holes that they chew through the cuticle of the host. Although chemicals serve as the main short term control agents, the compatibility of biological and chemical control has never been investigated for this system. This study examined the responses of immature and adult B. longissima and its larval parasitoid A. hispinarum to avermectin and acetamiprid. Avermectin caused complete mortality of 2nd to 4th instar larvae, and of adults of B. longissima at 10, 15 and 2 d after treatment, respectively. However, 26.7% of the 2nd instar larvae, 55.3% of the 4th instar larvae, and 74%, of adult B. longissima were still alive 40 d after acetamiprid application. Following avermectin exposure, 17.5%, 9.2% and 23% of mummified B. longissima larvae contained viable adult parasitoids for the parasitoid egg, larva and pupa treatments, respectively, and the numbers of dead parasitoids per mummy were 3.3, 7.2 and 13.3 for the egg, larva and adult treatments, respectively. However, for acetamiprid treatment, 70–75.9% of mummified B. longissima larvae contained viable adult parasitoids in all three stage treatments, and the number of dead parasitoids per mummy was 2.8, 2 and 3.4 in egg, larva and adult treatments, respectively. This study showed that a sublethal dose of avermectin is more toxic than acetamiprid to B. longissima and A. hispinarum. Therefore, direct contact of the parasitoid with avermectin should be avoided when this insecticide is used to control B. longissima.     

Dynamic analysis of gene expression and determination of chemicals in agarwood in Aquilaria sinensis

Agarwood is the resinous heartwood of Aquilaria species. However, low yields and high costs of existing stimulation methods have led to the need for new techniques to produce agarwood rapidly and effectively.We developed a biological agarwood-inducing technique(Agar-Bit) that produces high yields and quality within 6 months. To better understand agarwood formation by Agar-Bit, dynamic gene expressions of key synthetases pathways of sesquiterpenes and chalcone-related enzymes at different times were determined after both Agar-Bit and the traditional burning chisel drilling(BCD) stimulation on Aquilaria sinensis trees. The q RT-PCR results show that some characteristic synthase genes were expressed at greatly different levels and times compared with thecontrols. For the Agar-Bit technology, main changes were after the 3 rd or 5 th month, while BCD expression clearly changed at the 5 th month. Essential oils and total chromone contents were simultaneously determined. In the Agar-Bit group, both were higher and similar to natural levels. The Agar-Bit methodology is a new option for producing agarwood as demonstrated by genetic and chemical aspects. The differences in gene expression within 6 months for both groups indicates that the mechanisms of the two methods are different. These findings provide information on genetic variation during the process of agarwood formation.     

基于四环素调控系统构建白蛋白启动子调控大鼠uPA转基因表达的慢病毒载体

基于四环素调控系统构建白蛋白(albumin,Alb)启动子调控大鼠uPA(urokinase-type plasminogen activator,ruPA)转基因肝特异性过表达的慢病毒载体pLVX-Alb-TetOne-TRE-ruPA-T2A-CopGFP(pLATTRUTG)。以CTG0875-2-11质粒为模板,PCR扩增大鼠的uPA(ruPA)基因,3’端添加Flag标签,In-Fusion克隆至pLVX-Alb-TetOne-TRE-T2A-CopGFP(pLATTTG)质粒中,得到慢病毒载体pLVX-Alb-TetOne-TRE-ruPA-T2A-CopGFP(pLATTRUTG),所构建质粒经测序和酶切鉴定。将pLATTRUTG瞬时转染293T细胞,转染后24 h倒置荧光显微镜检测CopGFP表达;接着向6孔细胞培养板内加入强力霉素(Doxycycline,Dox),48 h后在倒置荧光显微镜下观察CopGFP表达(包括未加Dox的孔),随后收集细胞以提取总RNA和总蛋白,用于RT-qPCR检测ruPA及报告基因表达和Western blot检测标签蛋白Flag表达。酶切和测序确证我们成功构建了慢病毒载体pLATTRUTG;瞬转293T细胞后,24 h倒置荧光显微镜下可见零星细胞(约占0.1%)发弱的绿色荧光,加Dox 48 h后所有细胞展现强的绿色荧光,而不加Dox的孔内仍然只见到零星细胞(约占0.1%)发弱的绿色荧光。RT-qPCR和Western blot检测结果显示,与不加Dox的细胞相比,加Dox的细胞中ruPA、报告基因CopGFP和Flag表达水平显著升高。结果提示,成功基于四环素调控系统构建Alb启动子调控大鼠uPA转基因表达的慢病毒载体pLATTRUTG,为相关后续实验奠定了基础。     

Construction of recombinant retroviral vector of short interfering RNAs specific for macrophage migration inhibitory factor (MIF) and establishment of stable HeLa cell line with a persistent knockdown of MIF

AIM: To construct recombinant retroviral vector of short interfering RNAs (siRNA) specific for macrophage migration inhibitory factor (MIF) and to establish the stable knockdown of MIF cell line of mammalian cells by transfecting the recombinant retroviral vectors. METHODS: We synthesized oligo-nucleotides for MIF in vitro, and cloned them into retroviral vector pSuper.retro. Subsequently the plasmids were sequenced and digested to identify the construction of the recombinant retroviral vectors. The vectors RNAi were transfected into packing cell line PHOENIX, which was selected by puromycin later. HeLa cell line was infected by the virus supernatant of stable PHOENIX cell lines, and the stable HeLa cell line showed significantly to silence MIF was established by selecting with puromycin. We also compare the characters of HeLa-pSuper-mock to HeLa-pSuper-MIF cells by using migration assay, adhesion assay, soft agar assay and FACS analysis of the cell-cycle progression. RESULTS: The recombinant retroviral vectors were constructed successfully. The HeLa cell line infected by the supernatant containing the retrovirus of package PHOENIX cells was persistent knockdown of MIF confirmed by Western blotting. Knockdown of MIF in HeLa cells inhibited the migration and adhesion, and decreased the clone formation. FACS analysis revealed that knockdown of MIF arrested HeLa cells in G0/G1 phase. CONCLUSION: We establish the stable HeLa cell line with a persistent knockdown of MIF. Our current studies reveal that MIF is necessary for HeLa cell migration and anchorage-independent growth.     

Effect of breviscapin on Ito and IK1 channel current in isolated ventricular myocytes of rats

AIM: The effects of breviscapin on transient outward potassium current (I_to) and inward rectifier potassium current (I_K1) channel in isolated ventricular myocytes of rats were determined. The mechanism of breviscapin at the ionic channel level was explored. METHODS: Single ventricular myocyte of rats was isolated by enzymatic dissociation. The whole-cell patch-clamp recording technique was used to record the change of I_to and I_K1 channel current influenced by breviscapin. RESULTS: Breviscapin decreased the I_to channel current in a dose-dependent and voltage-dependent manner in ventricular myocytes of rats. (1) The current-voltage curve was significantly decreased. Breviscapin at concentrations of 0.02, 0.05, 0.08, 0.10 g·L^-1 respectively decreased I_to current density (10.07±0.50)%, (27.47±2.36)%, (42.72%±2.50)% and (56.09±5.60)%. I_to was inhibited in a voltage-dependent manner, showing a significant attenuating effect at test potentials from 0 to + 50 mV. (2) The I_to activation curve, the activation curve and the recovery curve were not altered. (3) Breviscapin did not affect I_K1. CONCLUSION: Breviscapin concentration-dependently decreases I_to channel current in ventricular myocytes of rat.