CRISPR/Cas9介导的多基因编辑系统在大豆中的应用

自CRISPR/Cas9（clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9）基因 组编辑技术发现以来，迅速在作物中得到广泛应用。但是，CRISPR/Cas9多基因编辑系统在大豆中的研究尚待开 发。本文利用CRISPR/Cas9介导的多基因编辑系统，分别构建了两个载体，一个载体含6个靶点，编辑7个大豆基因 （4个Glycine max ASYMMETRIC LEAVES1（GmAS1）同源基因和3个GmAS2 同源基因），另一个载体含8个靶点，编辑 11个G. max AGAMOUS 家族同源基因（4个GmAG 同源基因，2个G. max SEEDSTICK（GmSTK）同源基因和5个G. max SHATTERPROOF1（GmSHP1/2）同源基因）。大豆遗传转化后，经表型鉴定和靶点检测发现，CRISPR/Cas9介导的多 基因编辑系统在大豆中成功实现了多基因编辑。当3个GmAS1 同源基因和3个GmAS2 同源基因同时突变时，导致 大豆叶片向远轴面弯曲、皱缩且叶柄变短的表型。当2个GmSHP1 同源基因和2个GmSTK 同源基因同时突变时，导 致豆荚停止发育的不育表型。     

CRISPR/Cas9技术敲除酿酒酵母gpd2基因对产2,3-丁二醇的影响

旨在利用CRISPR/Cas9基因编辑技术敲除酿酒酵母甘油-3-磷酸脱氢酶基因(gpd2),探究其对2,3-丁二醇产量的影响。根据酿酒酵母(Saccharomyces cerevisiae)W5甘油-3-磷酸脱氢酶基因(gpd2)设计供体片段及gRNA片段,将gRNA片段与可表达Cas9蛋白的敲除载体相连,之后将重组质粒及供体DNA片段转化到S. cerevisiae W5细胞中,根据表型筛选及PCR验证获得gpd2基因缺失菌株。结果表明目的基因gpd2敲除成功,基因缺失菌株与原始菌株经发酵实验相比,甘油产量下降22.01%,乙醇产量提高24.65%,2,3-丁二醇产量下降10.60%。gpd2基因的敲除并没有提高2,3-丁二醇的产量,原因可能是逐渐积累的NADH会优先被细胞内大量的乙醇脱氢酶所氧化,作用于乙醇的产生,而不是优先作用于2,3-丁二醇的合成。本实验构建了适用于酿酒酵母的基因敲除系统,该系统对进一步探究酿酒酵母其他代谢产物与2,3-丁二醇合成之间的关系具有实际的借鉴意义。     

大鲵幼体蛋白质的需求量

为评定大鲵幼体对饲料蛋白质的需求量,以鱼粉为主要蛋白源配制6种蛋白质水平(干样基础)的实验饲料:D1(43.7%)、D2(47.1%)、D3(51.3%)、D4(55.7%)、D5(59.9%)和D6(64.4%),饲喂初始体质量为(20.99±0.15)g的大鲵幼体92 d。结果显示,①饲料蛋白质水平对大鲵增重率有显著影响,在D4组达到最大值,较D1组增加了276.4%,且全鲵蛋白质沉积率和肌肉RNA、RNA/DNA值、胃蛋白酶、H^+-K^+-ATPase、胰蛋白酶、脂肪酶和Na^+-K^+-ATPase、肝脏超氧化物歧化酶(SOD)均在D4组达到最佳,而肝脏和肠道丙二醛(MDA)在该组均达到最低;②随饲料蛋白质水平增加,肌肉粗蛋白线性增加,全鲵脂肪线性下降,全鲵水分和粗灰分在各组间差异不显著,全鲵粗蛋白先增加后趋于稳定,在D4组达到最大;③大鲵皮肤胶原蛋白含量在D4组达到最高,较D1组增加了27.83%。研究表明,以增重率、肌肉RNA/DNA值、蛋白质沉积率和皮肤胶原蛋白含量为评价指标,通过二次回归方程分析得到大鲵幼体饲料的最适蛋白质水平为55.9%~58.3%(干样基础),该饲料蛋白质水平能显著提高大鲵幼体胃的泌酸能力、机体消化吸收和抗氧化能力,增加鲵体营养素的沉积,从而促进生长和饲料的转化;而低蛋白质水平饲料显著抑制大鲵的生长。     

β-carotene attenuates weaning-induced apoptosis via inhibition of PERK-CHOP and IRE1-JNK/p38 MAPK signalling pathways in piglet jejunum

Weaning may cause oxidative injury, immune response impairment, apoptosis and other injuries in piglets. Oxidative and endoplasmic reticulum stress (ERS) can elicit inflammatory responses, and persistent oxidative and ERS also may lead to apoptotic cascades, which is associated with the pathogenesis of multiple diseases. β-carotene, a natural carotenoid, has potential anti-inflammatory and antioxidant functions. However, the effect of β-carotene on apoptosis in weaned piglets and the detailed molecular mechanism remain unclear. In this study, we found that β-carotene decreased malondialdehyde (MDA) levels and increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in piglet serum. β-carotene could inhibit the mRNA levels of caspase-3 significantly, but had no significant inhibitory effect of the mRNA levels of caspase-9 and caspase-12 in the piglet jejunum. In addition, β-carotene decreased the activation of GRP78, CHOP, and JNK/p38 MAPK and the ratio of Bax/Bcl-2. Furthermore, β-carotene had a significant influence on the activation of ERS and apoptosis-related signals in TG-induced IPEC-J2. In the present study, β-carotene pre-treatment attenuated the ratio of Bax/Bcl-2 and prevented TG-induced increases in the level of PERK-CHOP and IRE1-JNK/p38 MAPK pathway activation in a dose-dependent manner. Overall, these findings indicate that β-carotene may protect weaning-induced apoptosis through inhibiting ERS.     

利用CRISPR/Cas9系统编辑日本结缕草ZjSGR基因技术研究

日本结缕草(Zoysia japonica)是一种常见的具有众多优良性状的暖季型草种,秋冬季节在我国北方地区会较早出现叶片脱绿现象,在一定程度上限制了日本结缕草在北方的大面积使用。ZjSGR是从结缕草中分离出来的一种衰老诱导基因,负调控叶片滞绿性状。本研究利用CRISPR/Cas9系统,通过基因枪介导的遗传转化方法实现对日本结缕草ZjSGR基因的编辑,经测序验证获得1株单碱基缺失突变植株。突变植株叶片深绿,在正常生长温度(28~30℃)条件下,和对照相比,叶绿素含量更高(P<0.05)。本研究为进一步阐述ZjSGR基因在日本结缕草滞绿性状调控中的作用机理提供了基础研究材料,也为培育日本结缕草滞绿品种育种工作奠定了基础。     

CD36/Src/ERK pathway is involved in process of monocyte differentiation into macrophages

AIM To investigate the role of fatty acid translocase (FAT/CD36) on differentiation of monocytes to macrophages. METHODS Human monocyte THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA) at 0, 100 and 200 μg /L. Small interfering RNA (siRNA) targeting CD36 (siCD36) was employed to knock down the expression of CD36 in THP-1 cells. The CD36 over-expression (CD36OE) cell line was constructed by transfection with a recombinant lentivirus containing CD36 cDNA. Optical microscopy and crystal violet staining were used to detect the monocyte morphological changes and adhesion ability. The protein expression of CD36 was measured by flow cytometry and Western blot. The mRNA levels of CD36, CD11b and CD80 were detected by real-time PCR. The protein levels of extracellular signal-regulated kinase (ERK） and Src tyrosine kinase were determined by Western blot. RESULTS The cellular adhesiveness of THP-1 cells was elevated in the process of monocytes differentiation, and the expression of CD36 was increased in this process as well (P<0.01). siCD36 was transfected into the THP-1 cells (CD36i group) and the silencing efficiency was approximately 80%. The cell surface area and cellular adhesiveness were significantly decreased in CD36i group compared with scrambled siRNA (NCi) group (P<0.01). The mRNA levels of CD11b and CD80 were decreased in CD36i group compared with NCi group (P<0.01). The cell surface area and cellular adhesiveness were increased in CD36OE group compared with empty vector (vector) group (P<0.05). The mRNA levels of CD11b and CD80 were increased in CD36OE group compared with vector group (P<0.01). The phosphorylation levels of ERK and Src were decreased in CD36i group compared with NCi group (P<0.05). CONCLUSION CD36 promotes the differentiation of human monocyte THP-1 cells to macrophages by increasing the phosphorylation of Src and further activating ERK.     

Hyperbaric oxygen therapy activates CREB/BDNF signaling pathway to reduce synaptic damage in hippocampal neurons of Alzheimer disease mice

AIM To investigate the effect of hyperbaric oxygen (HBO) on synaptic damage of hippocampal neurons in APP/PS1 transgenic (TG) mice and its possible mechanism. METHODS The 6-month-old male APP/PS1 TG mice were randomly divided into TG group, HBO group and cAMP response element binding protein (CREB) inhibitor H89 group, with 10 mice in each group. Ten male wild-type (WT) C57BL/6 mice of the same age were used as negative control group (WT group). The mice in HBO and H89 groups were treated with HBO for 6 cycles, while the mice in WT group and TG group were not treated. The learning and memory abilities were observed by Morris water maze. The nesting ability of the mice was detected by nesting test. The Nissl bodies in hippocampal neurons were observed by Nissl staining. The mRNA expression of CREB and brain-derived neurotrophic factor (BDNF) in hippocampus was detected by real-time PCR. The protein levels of synapsin (SYN), postsynaptic density protein 95 (PSD95), growth-associated protein 43 (GAP43), CREB, phosphorylated CREB (p-CREB) and BDNF in the hippocampus were determined by Western blot. RESULTS Compared with WT group, the learning and memory abilities of the mice in TG group were signilficantly reduced (P<0.05). In addition, the nesting score, the number of Nissl bodies in the hippocampal neurons, the mRNA expression of CREB and BDNF, and the protein levels of SYN, PSD95, GAP43, p-CREB and BDNF were also decreased significantly (P<0.05). Compared with TG group, the learning and memory abilities of the mice in HBO group were improved (P<0.05). Meanwhile, the nesting scores of the mice were significantly increased (P<0.05), the neurons in the hippocampus were arranged neatly, and the number of Nissl bodies, the relative mRNA expression of CREB and BDNF,and the protein levels of SYN, PSD95, GAP43, p-CREB and BDNF were also increased significantly (P<0.05). Compared with HBO group, the mice in H89 group had poor learning and memory abilities, lowered nesting scores and decreased number of Nissl bodies. Futhermore, the relative mRNA expression of CREB and BDNF, and the protein levels of SYN, PSD95, GAP43, p-CREB and BDNF were also decreased significantly (P<0.05). CONCLUSION HBO improves the learning and memory abilities of APP/PS1 TG mice, and its mechanism may be related to activating the CREB/BDNF signaling pathway to reduce synaptic damage of hippocampal neurons in mice.     

Effects of platelet-rich plasma on chondrocyte apoptosis and NLRP3/IL-1β signaling pathway in rabbits with osteoarthritis

AIM To explore the effect of platelet-rich plasma (PRP) on rabbit osteoarthritis and its possible mechanism. METHODS The rabbits with knee osteoarthritis were prepared and then divided into model group, sodium hyaluronate (SH) group and PRP group, and another sham operation group was set up, with 6 rabbits in each group. The gross morphological changes of rabbit cartilage were observed. HE staining was used to evaluate the pathomorphological changes of the cartilage. TUNEL staining was used to detect the apoptosis of chondrocytes. The expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/interleukin-1β （IL-1β） signaling pathway-related molecules was observed by immunohistochemical staining, and the protein levels of caspase-3, Bcl-2 and Bax were determined by Western blot. Chondrocytes were isolated and processed according to grouping, and the NLRP3 and IL-1β levels of the cells were measured by ELISA. RESULTS Compared with sham operation group, Pelletier score, Mankin score, chondrocyte apoptotic rate, the positive protein expression rates of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax in model group were increased significantly (P<0.05), while the protein expression of Bcl-2 was decreased significantly (P<0.05). Compared with model group, Pelletier score, Mankin score, the apoptotic rate of chondrocytes, the positive protein expression rates of NLRP3, ASC, caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax in SH group and PRP group were decreased significantly (P<0.05), while the protein expression of Bcl-2 was increased significantly (P<0.05). In PRP group, Pelletier score, Mankin score, the apoptotic rate of chondrocytes, the positive protein expression rates of NLRP3, ASC, caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax were lower than those in SH group, while the protein expression of Bcl-2 was higher than that in SH group (P<0.05). Compared with control group, the expression of NL?RP3 and IL-1β in MCC950 (NLRP3 ihibitor) group were significantly reduced (P<0.05), the expression of NLRP3 in eucalyptol (IL-1β inhibitor) group was not significantly changed (P>0.05), and the expression of IL-1β was significantly reduced (P<0.05). CONCLUSION Platelet-rich plasma promotes the repair of cartilage in osteoarthritis rabbits, which has better effect than SH. The mechanism may be related to the inhibition of NLRP3/IL-1β pathway and the reduction of chondrocyte apoptosis.     

Panax notoginseng saponins suppress oxygen-glucose deprivation/reoxygenation-induced pyroptosis of SH-SY5Y cells

AIM To investigate the effect of Panax notoginseng saponins (PNS) on pyroptosis of SH-SY5Y cells induced by oxygen-glucose deprivation/reoxygenation (OGD/R). METHODS The OGD/R was conducted to induce ischemia/reperfusion injury in SH-SY5Y cells. The effects of PNS on the viability (detected by CCK-8 assay) and membrane permeability [indicated by lactate dehydrogenase (LDH) leakage and propidium iodide (PI) staining positive cell proportion] of OGD/R-induced SH-SY5Y cells were observed. The protein levels of gasdermin D (GSDMD), GSDMD N-terminal fragment (GSDMD-N), caspase-1 and caspase-4, and the release of interleukin-1β (IL-1β) and IL-18 in the cells were also determined. RESULTS After exposure to OGD/R, the viability of SH-SY5Y cells dramatically decreased (P<0.01), while the LDH leakage, the PI staining positive cell proportion, the protein levels of GSDMD, GSDMD-N, caspase-1 and caspase-4, and the release of IL-1β and IL-18 were significantly increased (P<0.01). However, PNS treatment enhanced the viability of SH-SY5Y cells inhibited by OGD/R (P<0.01), but reduced the leakage of LDH and the percentage of PI staining positive cells (P<0.05 or P<0.01). Moreover, PNS reversed the increases in the protein levels of GSDMD, GSDMD-N, caspase-1 and caspase-4 and the release of IL-1β and IL-18 in OGD/R-induced SH-SY5Y cells (P<0.05 or P<0.01). CONCLUSION Treatment with PNS alleviates OGD/R-induced injury in SH-SY5Y cells. Its mechanism may be related to inhibition of SH-SY5Y cell pyroptosis induced by OGD/R.     

Down-regulation of miR-153-3p attenuates H2O2-induced H9C2 cell injury by promoting Nrf2 expression

AIM To study the effect of microRNA-153-3p (miR-153-3p) knock-down on oxidative injury of H9C2 cells induced by H₂O₂ and its specific mechanism. METHODS The oxidative stress injury of H9C2 cell model was induced by H₂O₂, and then the cell viability and the expression of miR-153-3p were detected by MTT assay and RT-qPCR, respectively. The effects of miR-153-3p knock-down on the H9C2 cell injury under oxidative stress were studied by RNA interference technology. The targets of miR-153-3p were identified by Western blot and dual-luciferase reporter assay. RESULTS MTT assay showed that the viability of H9C2 cells was decreased with the increase in H₂O₂ concentration (P<0.05). The results of RT-qPCR showed that the expression of miR-153-3p was increased with the increase in H₂O₂ concentration (P<0.05). Knock-down of miR-153-3p increased the viability of H9C2 cells under oxidative stress, decreased the cell apoptosis and the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD). The expression of nuclear factor E2-related factor 2（Nrf2） and antioxidant response element（ARE） activity were increased with the increase in H₂O₂ concentration (P<0.01). TargetScan analysis and dual-luciferase reporter assay showed that Nrf2 was one of the potential target genes of miR-153-3p. The results of Western blot further showed that over-expression of miR-153-3p inhibited the expression of Nrf2 (P<0.01), while down-regulation of miR-153-3p increased the expression of Nrf2 (P<0.01). Dual interference with Nrf2 and miR-153-3p significantly reduced H9C2 cell viability， promoted the apoptosis, increased MDA content, and decreased SOD activity in the presence of H₂O₂ (P<0.01). CONCLUSION Inhibition of miR-153-3p expression attenuates the injury of H9C2 cells induced by H₂O₂ through up-regulating Nrf2/ARE signaling pathway.