不同施氮水平下灌浆期高温对水稻贮藏蛋白积累及其合成代谢影响

【目的】 揭示不同施氮水平下灌浆结实期高温对稻米贮藏蛋白积累及其组分的影响,明确不同温氮处理组合下稻米贮藏蛋白合成积累过程与籽粒氮代谢关键酶及相关基因表达间关系。 【方法】 以2个主栽常规晚粳品种（秀水134和秀水09）为材料,利用盆栽土培试验,在水稻穗分化期设低氮（每盆0.5 g尿素）和高氮（每盆2.0 g尿素）2个氮水平,继而通过在水稻灌浆结实期的人工气候箱控温试验,设置高温（日均温度30℃,日最高温和最低温分别为34℃和26℃）和常温（日均温度23℃,日最高温和最低温分别为26℃和20℃）,构成低氮-常温（LN-NT）、低氮-高温（LN-HT）、高氮-常温（HN-NT）、高氮-高温（HN-HT）4个处理组合,并结合水稻籽粒灌浆不同时期取样,探讨氮素穗肥对水稻高温灌浆过程贮藏蛋白积累和主要蛋白组分含量影响及其与籽粒氮代谢关键酶及相关基因表达间关系。 【结果】 氮素穗肥和灌浆期高温均会导致稻米粗蛋白相对含量上升,但在高温处理下单位籽粒中粗蛋白的绝对量却呈降低趋势,以13 kD醇溶蛋白亚基组分在高温处理下的下降幅度最大,从而引起籽粒谷蛋白/醇溶蛋白质比值的上升,其原因主要是由于编码水稻13 kD醇溶蛋白合成基因（Pro13, Pro14和Pro17）在高温处理下的下调表达所致。与之相比,增施氮素穗肥（HN-NT和HN-HT）在引起稻米粗蛋白相对含量提升的同时,单位籽粒中的粗蛋白总量、谷蛋白和醇溶蛋白含量均显著增加,但对贮藏蛋白谷/醇比的影响不明显,且谷蛋白组分中的37 kD酸性亚基和22 kD碱性亚基在不同氮处理水平下的相对比例也基本保持稳定;氮素穗肥可增强灌浆籽粒中的谷氨酰胺合酶（GS）、谷草转氨酶（GOT）和谷丙转氨酶（GPT）活性,但HN-HT处理下的籽粒GS、GOT和GPT活性显著低于HN-NT处理,高温胁迫对水稻灌浆中后期籽粒器官中的氮转运代谢具有抑制作用。此外,在不同氮素水平下,灌浆期高温均可引起稻米整精米率的明显下降和垩白度的显著上升,但HN-HT处理的千粒重、结实率、产量水平、整精米率却高于LN-HT处理,且前者的稻米垩白度显著低于后者,氮素穗肥不足加剧了高温胁迫对千粒重、结实率、整精米率和垩白度等产量和品质性状的负面影响。 【结论】 氮素穗肥对水稻高温灌浆过程贮藏蛋白合成积累起重要调节作用,增施氮素穗肥虽然对水稻籽粒灌浆过程中的谷蛋白和醇溶蛋白质合成具有促进作用,并导致稻米贮藏蛋白相对量与绝对量增加,但对于高温灌浆下13kD醇溶蛋白亚基合成及其组分含量下降具有一定程度的缓解效应,有利于维持贮藏蛋白谷/醇比相对稳定。     

Variation in glomalin in soil profiles and its association with climatic conditions,shelterbelt characteristics,and soil properties in poplar shelterbelts of Northeast China

Glomalin-related soil protein(GRSP)sequesters large amounts of carbon and plays important roles in maintaining terrestrial soil ecosystem functions and ecological restoration;however,little is known about GRSP variation in 1-m soil profiles and its association with stand characteristics,soil properties,and climatic conditions,hindering GRSP-related degraded soil improvement and GRSP evaluation.In this study,we sampled soils from 1-m profiles from poplar(Populus spp.)shelterbelts in Northeast China.GRSP contents were 1.8–2.0 times higher in the upper 40 cm soil layers than at 40–100 cm.GRSP-related soil organic carbon(SOC)sequestration in deeper soil layers was*1.2 times higher than in surface layers.The amounts of GRSP-related nutrients were similar throughout the soil profile.A redundancy analysis showed that in both surface and deeper layers,soil properties(pH,electrical conductivity,water,SOC,and soil nutrients)explained the majority of the GRSP variation(59.5–84.2%);the second-most-important factor in GRSP regulation was climatic conditions(temperature,precipitation,and altitude),while specific shelterbelt characteristics had negligible effects(<5%).Soil depth and climate indirectly affected GRSP features via soil properties,as manifested by structural equation model analysis.Our findings demonstrate that GRSP is important for carbon storage in deep soils,regardless of shelterbelt characteristics.Future glomalin assessments should consider these vertical patterns and possible regulating mechanisms that are related to soil properties and climatic changes.     

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。     

长期秸秆还田和地下水位对土壤镉积累及有效性的影响

以1982年开始的长期定位试验为对象,研究了2个地下水位(高水位-20 cm和低水位-80 cm)下3种施肥方式(高量秸秆还田HS、常量秸秆还田MS、化肥CF)对水稻土镉积累及有效性的影响。结果表明:高水位条件下,长期秸秆还田可增加土壤总镉及有效态镉含量,HS和MS处理分别较CF处理提高21.8%、59.9%和9.8%、49.2%。低水位条件下,HS处理土壤总镉及有效态镉含量高于CF处理,较CF处理分别提高11.2%和31.6%; MS处理土壤总镉及有效态镉含量低于CF处理,较CF处理分别下降8.8%和14.3%。2012年,在保证原定位试验有足够重复的前提下,变更高水位条件下的部分试验处理。当HS处理变更为CF处理5 a后,土壤总镉和有效态镉含量分别较变更前下降2.5%和5.7%;CF处理变更为MS处理后,土壤总镉和有效态镉含量分别较变更前增加16.5%和58.9%。相同施肥处理,高水位土壤总镉含量高于低水位土壤。在MS处理下,高水位土壤有效态镉含量也高于低水位土壤,而在HS和CF处理下,低水位土壤有效态镉含量高于高水位土壤。土壤镉含量和土壤基本理化性质的相关分析表明,土壤总镉与土壤有机质、络合态铁含量极显著正相关,与pH显著负相关;土壤有效态镉与土壤pH、游离氧化铁极显著负相关。研究表明,施肥方式和地下水位是通过改变土壤的基本性质影响土壤镉的积累及其有效形态。     

长江下游农区金花菜叶蛋白提取工艺优化及关键参数特征

为改进和优化金花菜(Medicago polymorpha)叶蛋白提取的工艺环节,并提高叶蛋白提取率及提取物纯度等参数指标,本研究选择长江下游农区广泛种植的金花菜为原料,通过设置较为合理科学的料液比、提取温度、加热时间、pH以及提取剂(酸)种类进行叶蛋白提取单因素试验。结果表明:各单因素试验最佳参数分别为料液比1∶3、提取温度70℃、加热时间15 min、pH 4、提取剂(酸)为盐酸。基于清洁安全生产角度,选择了单因素试验中提取指标同样较高的柠檬酸作为提取剂,设置了料液比、pH及提取温度的3因素3水平响应面试验,发现影响叶蛋白提取率的因素依次为pH、提取温度、料液比。得到最佳提取工艺:料液比1∶3、pH 4.1、提取温度72℃,相应的叶蛋白提取率为49.63%,提取物纯度为56.87%。本研究优化的技术工艺及相关参数指标可为农区豆科草类植物多元化利用及叶蛋白工业化生产提供一定的技术支撑和相关借鉴。     

Effects of calpain-2 and autophagy-related protein 5 on hepatocyte apoptosis induced by endoplasmic reticulum stress

AIMTo investigate the effects of calpain-2 and autophagy-related protein 5 (Atg5) on apoptosis of BRL-3A rat normal liver cells during endoplasmic reticulum stress (ERS) induced by dithiothreitol (DTT). METH?ODS: BRL-3A cells were treated with DTT at 2.0 mmol/L for 0, 6, 12 and 24 h to induce ERS. Real-time cell analysis (RTCA) was used to measure the effect of DTT on BRL-3A cell proliferation. Apoptosis and cell cycle distribution were analyzed by flow cytometry. The mRNA expression of calpain-2 and Atg5 was detected by real-time PCR. The protein levels of calpain-2, Atg5, Atg7, Atg12 and microtubule-associated protein 1 light chain 3 (LC3) were determined by Western blot. The interaction between calpain-2 and Atg5 was investigated by co-immunoprecipitation (Co-IP). RESULTSThe proliferation of BRL-3A cells treated with DTT was significantly inhibited. The apoptosis of BRL-3A cells was significantly increased after DTT treatment for 6, 12 and 24 h as compared with 0 h group (P<0.05). The cell cycle was arrested in G₁ phase after DTT treatment (P<0.05). After DTT treatment for 6, 12 and 24 h, the mRNA expression of calpain-2 and Atg5 in the BRL-3A cells was significantly increased as compared with 0 h group (P<0.05). The protein levels of calpain-2, Atg12 and Atg7 in the cells treated with DTT for 6, 12 and 24 h were significantly higher than those in 0 h group, and the ratio of LC3-II/LC3-I was also significantly higher than that in 0 h group, while Atg5 expression was significantly lower than that in 0 h group (P<0.05). The results of Co-IP found that the anti-calpain-2 antibody precipitated Atg5 protein from the cell lysates, and the anti-Atg5 antibody also precipitated calpain-2 from the cell lysates, which confirmed the interaction between calpain-2 and Atg5. CONCLUSION Calpain-2 may participate in ERS-induced hepatocyte apoptosis by interacting with Atg5.     

mTOR kinase inhibitor CC-223 suppresses human breast cancer cell viability

AIM: To investigate the inhibitory effect of CC-223, an inhibitor of mammalian target of rapamycin (mTOR) kinase, on the viability of human breast cancer cells and its mechanism. METHODS: The inhibitory effect of CC-223 on the viability of MCF-7 cells and MDA-MB-231 cells was measured by CCK-8 assay. The cell cycle distribution of breast cancer cells was examined by flow cytometry. The expression of cell cycle-related proteins and oncoproteins c-Myc and survivin was analyzed by Western blot. RESULTS: CC-223 significantly inhibited the viability of both MCF-7 and MDA-MB-231 cells (P<0.05). CC-223 induced cell cycle arrest in both G₁ phase and G₂/M phase in the MCF-7 cells (P<0.05). However, low concentration of CC-223 treatment resulted in the accumulation of MDA-MB-231 cell cycle in the G₂/M phase, and the cell number in G₁ phase was unaffected. Treatment with CC-223 for 24 h clearly inhibited the protein levels of cyclin B1, cyclin D1 and phosphorylated cell division cycle protein 2 in the breast cancer cells (P<0.05). CC-223 suppressed the expression of c-Myc and survivin in both MCF-7 cells and MDA-MB-231 cells (P<0.05). CONCLUSION: CC-223 inhibits cell viability by blocking cell cycle progression and down-regulating expression of c-Myc and survivin in both MCF-7 and MDA-MB-231 cells.     

马链球菌兽疫亚种烯醇化酶对小鼠肺泡巨噬细胞吞噬功能的影响

旨在研究马链球菌兽疫亚种（Streptococcus equi ssp.zooepidemicus，SEZ）烯醇化酶（enolase，Eno）对小鼠肺泡巨噬细胞（RAW264.7）吞噬能力的影响。通过构建原核表达质粒获得重组烯醇化酶（rEno），采用台盼蓝活细胞计数法，判定在不同处理浓度和时间下rEno蛋白对RAW264.7细胞的细胞毒性。将rEno蛋白与RAW264.7细胞共孵育后，用SEZ作用于细胞并检测细胞吞菌数量，判断RAW264.7细胞对SEZ的吞噬活性。进一步通过活细胞稳定同位素标记技术（SILAC）和蛋白质谱分析技术（LC-MS/MS），筛选到RAW264.7细胞中可能与SEZ Eno存在相互作用的候选蛋白。结果发现，10 μg·mL^-1 rEno蛋白处理对RAW264.7细胞有明显的细胞毒性，且10 μg·mL^-1 rEno蛋白处理RAW264.7细胞2和4 h可显著抑制其对SEZ的吞噬作用（P<0.01、P<0.05）。初步筛选到RAW264.7细胞中动力蛋白激活蛋白亚单位蛋白（dynactin subunit protein 2，Dctn）、整合素α-M蛋白（integrin alpha-M）等17种可能与Eno发生互作的蛋白。本研究获得了rEno重组表达蛋白，发现rEno可减少RAW264.7细胞对SEZ的吞噬，互作蛋白的初步筛选也为进一步揭示Eno在SEZ抗吞噬中的作用机制奠定了基础。     

与鸭C4结合蛋白互作的鸭疫里默菌外膜蛋白的筛选及鉴定

旨在筛选并鉴定与鸭C4结合蛋白（C4b-binding protein，C4BP）互作的鸭疫里默氏菌（Riemerella anatipestifer，R.anatipestifer）外膜蛋白。本研究将保存的R.anatipestifer复苏培养，提取外膜蛋白，以鸭C4BPα作为诱饵蛋白进行His pull-down及LC-MS/MS蛋白质谱鉴定，筛选与鸭C4BP可能发生互作的候选外膜蛋白；将各候选蛋白进行克隆、原核表达，免疫小鼠制备多克隆抗体，利用Far-western blot验证与鸭C4BP发生相互作用的R.anatipestifer外膜蛋白；针对候选蛋白及C4BPα各功能结构域进行克隆及原核表达，利用Far-western blot鉴定候选蛋白及与C4BP的相互作用位点；利用ELISA对补体因子C3b、C4b及C4BP在R.anatipestifer表面沉积情况进行测定，验证候选蛋白的功能。结果显示，经His pull-down及LC-MS/MS蛋白质谱分析，共筛选出3个与鸭C4BP发生相互作用的R.anatipestifer外膜蛋白，即ECE-1、SODs和Omp62；成功获得3个外膜蛋白多克隆抗体，ELISA检测3种多克隆抗体效价均超过1∶6 400，Western blot检测3种多克隆抗体可以与重组蛋白发生特异性反应；Far-western blot结果显示，仅ECE-1能够与C4BP发生相互作用，并且只有ECE-1全长能与鸭C4BP相互作用，而鸭C4BP与ECE-1的相互作用区域位于C4BPα的SCR 2和SCR 3；当健康鸭血清稀释度为3.125%时，ECE-1抗体能够显著促进补体因子C3b、C4b在R.anatipestifer表面的沉积作用（P<0.05），当健康鸭血清稀释度为6.25%时，ECE-1抗体能够显著抑制C4BP在R.anatipestifer表面的沉积作用（P<0.05）。本研究成功筛选并鉴定出1个与C4BP互作的R.anatipestifer外膜蛋白ECE-1，为进一步阐明R.anatipestifer免疫逃逸机制奠定了基础。     

基于S基因序列分析新型猪流行性腹泻病毒的遗传演化

最近，笔者实验室在青藏高原地区发现两种新亚型藏猪源猪流行性腹泻病毒（PEDV），为进一步调查新型PEDV是否在四川腹泻猪群中存在或流行，对实验室2018-2019年保存的116份猪腹泻粪便或肠组织样本进行PEDV的检测及其纤突蛋白基因（spike）分子特征研究。结果表明：腹泻样本的PEDV检出率为42.2%（49/116，95% CI=33.1%~51.8%），并获得了13条完整的S基因序列，全长为4 149~4 170 bp，序列相似性为94.2%~99.9%，其中SWUN-H3-CH-SCYA-2019的S基因与藏猪源新G1亚群PEDV的序列相似性高达97.0%~98.6%。遗传演化研究结果表明13株PEDV S基因划分为G1和G2大群，其中SWUN-H3-CH-SCYA-2019位于藏猪源新G1亚群；SWUN-19-CH-SCZY-2018、SWUN-4-CH-SCXC-2018、SWUN-1-CH-SCNJ-2019和SWUN-3CH-CH-SCZG-2019位于G2亚群中一个独立的分支，且与藏猪源新G2亚群毒株有着较近的亲缘关系。为了进一步研究13株PEDV的演化过程，以贝叶斯进化分析软件包（BEAST）进行分歧时间估算，结果表明SWUN-H3-CH-SCYA-2019的分歧时间约为2012.3年，早于藏猪源新G1亚群其余毒株的最早分歧时间（2015.7年）；SWUN-4-CH-SCXC-2018、SWUN-19-CH-SCZY-2018和SWUN-3CH-CH-SCZG-2019的分歧时间约为2014.2年，早于G2亚群的藏猪源毒株2014.7年，所有藏猪源PEDV的分歧时间均晚于四川毒株。本研究在四川地区首次发现了藏猪源PEDV，并且从毒株的分歧时间推断青藏高原的藏猪源PEDV来源于四川，为新型PEDV分子遗传进化的监测提供了依据。