人溶菌酶密码子优化及其在牛乳腺细胞中高效表达

【目的】分析牛乳腺中7种主要乳蛋白基因密码子使用偏好性,筛选高频密码子和低频密码子,依据其对人溶菌酶基因进行密码子局部优化和全局优化,通过牛乳腺上皮细胞等多种细胞对优化效果进行分析评价,为提高重组人溶菌酶表达量,开发新型、高效、安全的重组人溶菌酶提供理论依据。【方法】利用CodonW和EMBOSS等软件对7种主要牛乳蛋白和人溶菌酶基因密码子偏好进行生物信息学分析,筛选牛乳蛋白基因高频、低频密码子并根据牛乳蛋白基因密码子使用偏好对人溶菌酶基因翻译起始区前22位密码子（LYZop22）和全局密码子(LYZop)分别进行优化。构建人溶菌酶-荧光素酶融合表达载体（pGL3-LYZcw/op22/op）和人溶菌酶过表达载体（pcDNA-LYZcw/op22/op）,将上述载体分别转染牛乳腺上皮细胞(BMEC)、牛成纤维细胞（BFFC）和C127小鼠乳腺上皮细胞等3种细胞,通过荧光素酶、实时荧光定量PCR及Western-blot等方法检测密码子优化对溶菌酶表达的影响。【结果】牛乳蛋白基因密码子偏好以GC结尾,GC3s平均含量为0.537±0.062,而人溶菌酶GC3s含量为0.407,偏好以AT结尾;聚类结果表明,牛乳中酪蛋白与乳清蛋白类基因在密码子使用偏好性上也存在一定差异。依据筛选获得的牛主要乳蛋白基因5个高频密码子（RSCU>1.5）和7个低频密码子（RSCU<0.5）对人溶菌酶基因密码子进行优化并转染多种细胞进行表达效果分析。荧光素酶分析发现,相比野生型溶菌酶密码子（LYZcw）,翻译起始区密码子优化类型LYZop22分别在BMEC、BFFC细胞中提高1.48倍（P<0.01）和1.30倍（P>0.05）;而全局密码子优化类型LYZop分别在BMEC、BFFC中提高2.2倍（P<0.01）和2.44倍（P<0.01）,说明密码子优化能明显提高人溶菌酶在多种细胞中表达量。实时荧光定量PCR结果表明,LYZop22相比LYZcw在BMEC和BFFC细胞中分别提高了2.08倍（P<0.05）和1.5倍（P>0.05）,而LYZop则分别提高了22倍（P<0.01）和17.8倍（P<0.01）,mRNA表达水平与密码子优化后的mRNA二级结构稳定性呈正相关。Western-blot结果也进一步表明,密码子优化后的LYZop22和LYZop能明显提高重组人溶菌酶在牛乳腺上皮细胞中的表达量。上述结果表明,根据牛乳腺中主要乳蛋白基因密码子使用偏好进行密码子优化,能显著提高人溶菌酶在牛乳腺上皮细胞及成纤维细胞中的表达量,且溶菌酶基因全局密码子优化效果优于翻译起始区密码子优化效果。【结论】通过生物信息学分析获得了牛乳蛋白基因使密码子使用偏好及高低频密码子;依据牛乳蛋白密码子使用偏好性对人溶菌酶密码子优化能显著提高重组人溶菌酶mRNA水平和蛋白水平的表达量,为今后利用生物反应器高效生产重组人溶菌酶奠定基础。     

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.     

药剂拌种对春油菜白粉病和茎象甲的防效及对油菜产量的影响

为明确不同药剂拌种对春油菜出苗率、病虫害防效及产量的影响, 本文在3种不同种植密度下, 针对春油菜白粉病、油菜茎象甲, 选择25 g/L咯菌腈FSC、600 g/L吡虫啉SC、30%噻虫嗪SC、600 g/L吡虫啉SC+25 g/L咯菌腈FSC、30%噻虫嗪SC+25 g/L咯菌腈FSC进行了拌种处理试验。结果表明, 各拌种处理对春油菜的出苗有一定的影响, 其中600 g/L吡虫啉SC、30%噻虫嗪SC、600 g/L吡虫啉SC+25 g/L咯菌腈FSC、30% 噻虫嗪SC+25 g/L咯菌腈FSC拌种对春油菜出苗的影响大于25 g/L咯菌腈FSC拌种; 600 g/L吡虫啉SC+25 g/L咯菌腈FSC、30%噻虫嗪SC+25 g/L咯菌腈FSC拌种对白粉病和茎象甲的防效均高于单剂拌种; 600 g/L吡虫啉SC+25 g/L咯菌腈FSC拌种, 对春油菜白粉病的最高防效可达59.3%, 30%噻虫嗪SC+25 g/L咯菌腈FSC拌种对油菜茎象甲最高防效可达69.2%。白粉病病情指数与春油菜种植密度呈正相关, 拌种防效随密度增加而降低, 综合产量分析, 最佳种植密度为28.5万～34.5万株/hm2。     

A data transfer method for improving seed identification of maize (Zea mays) haploid breeding based on genetic similarity

Maize haploid breeding technology is able to identify haploid seeds non‐destructively, rapidly and at low cost with the help of Near‐infrared (NIR) spectral analysis. However, due to the hybridization of numerous parents and the low production rate of haploid, the haploid data collection becomes a burden for engineering this technology. Biologically, there are considerable similarities between the progeny of the same female parent and different male parents. Based on this advantage, similar spectral data can be transferred when the NIR technology is employed. A revised method of Transfer adaptive boost (TrAdaBoost) is proposed to improve identifying for the backpropagation neural network (BPNN) classifier. To avoid the negative transfer, a screening thresh is used to select out similar data, and the amount of these data are limited to join current training. The results show that the identification performances are improved significantly when the data amount is small. This method shows a high ability to make the seed identification more convenient for engineering maize haploid breeding.     

黔东南州打造全域森林公园的可行性研究

指出了建设全域森林公园是经济和社会发展的必然要求。在对黔东南州发展全域森林公园进行必要性和打造条件后认为:黔东南州在打造全域森林公园方面具有明显的政策优势,有着优良的森林生态环境资源,以及良好的林业产业基础等优势条件。通过不断完善相关的配套服务体系,已具备了打造全域森林公园的可行性。     

京津冀一次区域性暴雨特征分析

利用FNL再分析资料、卫星资料和自动站资料对2019年7月22~23日京津冀一次区域性暴雨过程进行了环流形势、卫星云图、动热力结构的分析。结果表明:此次暴雨发生在500hPa两槽一脊、副热带高压北抬、低空急流的大尺度环流背景下。西南方向的水汽输送和黄渤海的补充水汽为暴雨提供了良好的水汽条件,在河北中部的水汽辐合抬升则为暴雨提供了抬升条件。此次暴雨的产生受中尺度对流云团的连续生成合并影响,降水效率高。在暴雨中心整层表现为低层辐合、高层辐散,上升速度中心明显,使得高层的抽吸作用更强,更有利于上升运动的维持与发展,为降水强度增大提供了有力的动力条件,高空弱冷空气的入侵对能量锋区不稳定能量的释放起到了触发作用。     

红花双列杂交后代羟基黄色素A含量的遗传效应分析

选用6个羟基红花黄色素A（HSYA）含量差异较大的亲本,采用双列杂交方法配制杂交组合,测定2015年和2016年2年亲本及其后代F₁和F₂红花中的HSYA含量。运用双子叶植物种子数量性状遗传模型和统计分析方法,分析胚、细胞质和母体植株3套遗传体系的基因效应和环境互作效应。结果发现：在HSYA含量的遗传体系中,母体遗传效应影响最大,胚效应次之,细胞质效应影响最小。3套遗传体系均表现出基因主效应大于环境互作效应。机误方差较大,说明HSYA含量还受环境机误或抽样误差的影响。亲本遗传效应分析表明,豫红花1号（P₁）做亲本表现稳定,有利于增加杂交后代HSYA含量,达到提高品质、改良品种的效果。胚显性方差和母体显性方差均达到极显著水平,表明同时存在种子杂种优势和母体杂种优势,而且其主效应基因不受环境影响。综合考虑遗传主效应、胚显性效应和母体显性效应,亲本组合（P₁×P₅）有利于提高后代杂交品种的HSYA含量。该研究结果可为后代材料在杂种优势利用中的亲本选择提供理论支持。     

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.     

Effects of cardiac contractility modulation on ventricular electrical remodeling in rabbits with chronic heart failure

AIM: To investigate the effects of cardiac contractility modulation (CCM) on ventricular electrical remodeling in a rabbit model of chronic heart failure. METHODS: The rabbits were divided into sham group, heart failure(HF) group and HF+ CCM group. The rabbit model of chronic heart failure was established by ligating the ascending aortic root. Then electrical stimulations during the absolute refractory period were delivered lasting 6 h everyday for 4 weeks in rabbits of HF+ CCM group. The QTc and ventricular effective refrective period (VERP) were recorded. The protein and mRNA levels of Kv1.4, Kv4.3 and connexin 43 (Cx43) were determined by Western blot and RT-qPCR. RESULTS: Compared with sham group, QTc were significantly prolonged in HF rabbits at week 12 (P<0.05). CCM therapy shortened QTc of rabbits with heart failure at week 16 (P<0.05). Compared with sham group, VERP significantly increased in HF group and HF+ CCM group, while CCM therapy shortened VERP of rabbits with heart failure at week 16 (P<0.05). Compared with sham group, the mRNA and protein levels of Kv1.4, Kv4.3 and Cx43 were decreased in HF group and HF+ CCM group (P<0.05). However, CCM therapy restored the mRNA and protein levels of Kv1.4, Kv4.3 and Cx43 of rabbits with heart failure (P<0.05). CONCLUSION: CCM suppresses ventricular electrical remodeling in heart failure and the underlying mechanism may be associated with increasing Kv1.4, Kv4.3 and Cx43 expression.