miR-122靶向调控鸡BZW2基因的表达

miR-122(MicroRNA-122)是在肝脏中高表达的miRNA,在肝脏的生长发育和脂类代谢等过程发挥重要作用,BZW2 (basic leucine zipper and W2 domains 2)主要参与蛋白质合成代谢,本研究目的是确定BZW2是否为miR-122调控的靶基因.通过生物信息学预测miR-122的靶基因,并分析BZW2的3-UTR区域与miR-122种子区的配对情况,再用双荧光素酶报告系统和突变实验证明miR-122作用于BZW2的3'UTR.用荧光定量PCR检测转染miRNA-122mimic的鸡(Gallus gallus)肝癌细胞系(leghorn hepatocellar,LMH)细胞和转染LNA-antimiR-122的鸡原代肝细胞中BZW2的表达.鸡BZW2的3'-UTR区域与miR-122种子区互补配对,荧光素酶报告基因和突变实验分析表明,miR-122能够通过与BZW23'-UTR区结合抑制基因的表达;将miR-122在LMH细胞中过表达后,发现BZW2 mRNA表达水平显著下降;利用LNA-antimiR-122抑制鸡肝脏原代细胞中的miR-122后,BZW2 mRNA表达水平呈显著性上升.结果证明BZW2是miR-122的靶基因,其在mRNA水平上受到miR-122的负性调控,本研究为揭示miR-122在鸡肝脏中的广泛的功能提供了理论依据.     

Transgenic expression of a sorghum gene (SbLRR2) encoding a simple extracellular leucine-rich protein enhances resistance against necrotrophic pathogens in Arabidopsis

Plant leucine-rich repeat (LRR) domain-containing proteins are known to play important roles in signaling transduction and defense responses. In sorghum, SbLRR2 is pathogen-inducible gene encoding a simple extracellular LRR protein. Here, we demonstrated an earlier and stronger expression of SbLRR2 in a sorghum resistant genotype in comparison to a susceptible genotype following inoculation with the anthracnose pathogen (Colletotrichum sublineolum). In addition, SbLRR2 expression was found to be induced strongly by methyl-jasmonate treatment. Functional analysis was performed in SbLRR2 over-expression (OE) Arabidopsis plants, which showed enhanced resistance against the necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola. In addition, the OE lines were found to have elevated expression of several jasmonate acid (JA)-associated genes and higher endogenous JA contents. Hence, the SbLRR2-mediated defense responses in transgenic Arabidopsis are likely to be dependent on JA-signaling through increased JA production. On the other hand, the OE lines remained susceptible to Pseudomonas syringae pv. tomato like the wild type plants. Consistently, there was no up-regulation of salicylic acid (SA) defense marker gene expression or SA levels in the OE lines. Our results suggested that SbLRR2 is potentially useful for enhancing resistance against necrotrophic pathogens in transgenic dicot crops.     

玉米11个ZmbZIP基因的鉴定及表达特征分析

以玉米自交系郑58为试验材料,利用生物信息学方法和qPCR技术,设置200 mmol/L NaCl、20%PEG6000、4 ℃低温和硝态氮或铵态氮缺乏等胁迫试验,对11个ZmbZIP基因进行生物信息学分析,探测这些基因应答低温、干旱、盐害、缺氮等胁迫时的响应表达模式。系统进化树分析结果表明,11个ZmbZIP基因可以细分为2个亚组;qPCR分析结果表明,不同亚组的ZmbZIP基因在玉米不同组织中呈现不同的表达特征,同一亚组的基因呈现类似的表达模式,反映了ZmbZIP基因在玉米生长发育进程中生物学功能的保守性和分化性。人为模拟盐、干旱、低温和氮缺乏等胁迫的结果表明,ZmbZIP基因受到各种逆境因子的广泛调控,亚组I中的ZmbZIP1、ZmbZIP6和ZmbZIP13的表达量同时受NaCl溶液胁迫诱导而上调(相对表达量上调2倍),受PEG6000胁迫抑制下调;亚组II中的ZmbZIP38、ZmbZIP77、ZmbZIP112和ZmbZIP124同时受盐、PEG6000和低温胁迫诱导上调,硝态氮缺乏胁迫抑制ZmbZIP1、ZmbZIP6和ZmbZIP134基因表达,而铵态氮缺乏胁迫下,这3个基因表达明显上调,推测这些基因广泛参与应答逆境胁迫响应途径。     

Leucine markedly regulates pancreatic exocrine secretion in goats

Four goats (30.1 ± 1.3 kg) with common bile duct re‐entrant catheter and duodenal catheter were used to evaluate the effects of duodenal leucine infusion on pancreatic exocrine secretion and plasma parameters with two 4 × 4 Latin square design experiments. In the long‐term infusion experiment, goats were fed twice daily [700 g/day, dry matter (DM) basis] at 8:00 and 18:00 hours and were duodenally infused with 0, 3, 6, 9 g/day leucine for 14 days. Pancreatic juice and jugular blood samples were collected over 1‐h intervals for 6 h daily from d 11 to 14 days to encompass a 24‐h day. In the short‐term experiment, goats were infused leucine for 10 h continuously at the same infusion rate with Experiment 1 after feed deprivation for 24 h repeated every 10 days. Pancreatic juice and blood samples were collected at 0, 1, 2, 4, 6, 8 and 10 h of infusion. The results showed that the long‐term leucine infusion did not affect pancreatic juice secretion, protein output, trypsin and lipase secretion and plasma insulin concentration, but linearly increased α‐amylase secretion. No changes in pancreatic protein and lipase secretion were observed in the short‐term infusion. Pancreatic juice and α‐amylase secretion responded quadratically, with the greatest values observed in the 3 and 6 g/day leucine respectively. Trypsin secretion linearly decreased, while plasma insulin concentration increased linearly with increased leucine infusion. The results demonstrated that duodenal leucine infusion dose and time dependently regulated pancreatic enzyme secretion not associated with the change in plasma insulin concentration.     

亮氨酸调节哺乳动物骨骼肌蛋白质合成的研究进展

本文综述了亮氨酸处理对哺乳动物(主要是鼠和猪)骨骼肌及其细胞蛋白质合成的影响规律,并分析讨论了这一过程中亮氨酸的作用机制.近年来,大量的体内和体外试验研究表明,长期或短期高剂量的亮氨酸处理能够刺激哺乳动物骨骼肌内蛋白质的合成,而这不仅仅是由于亮氨酸可以为骨骼肌蛋白质合成提供能量和基质,还由于其可以调节骨骼肌细胞内与蛋白...     

Repeated post-exercise administration with a mixture of leucine and glucose alters the plasma amino acid profile in Standardbred trotters

Background

The branched chain amino acid leucine is a potent stimulator of insulin secretion. Used in combination with glucose it can increase the insulin response and the post exercise re-synthesis of glycogen in man. Decreased plasma amino acid concentrations have been reported after intravenous or per oral administration of leucine in man as well as after a single per oral dose in horses. In man, a negative correlation between the insulin response and the concentrations of isoleucine, valine and methionine have been shown but results from horses are lacking. This study aims to determine the effect of repeated per oral administration with a mixture of glucose and leucine on the free amino acid profile and the insulin response in horses after glycogen-depleting exercise.

Methods

In a crossover design, after a glycogen depleting exercise, twelve Standardbred trotters received either repeated oral boluses of glucose, 1 g/kg body weight (BW) at 0, 2 and 4 h with addition of leucine 0.1 g/kg BW at 0 and 4 h (GLU+LEU), or repeated boluses of water at 0, 2 and 4 h (CON). Blood samples for analysis of glucose, insulin and amino acid concentrations were collected prior to exercise and over a 6 h post-exercise period. A mixed model approach was used for the statistical analyses.

Results

Plasma leucine, isoleucine, valine, tyrosine and phenylalanine concentrations increased after exercise. Post-exercise serum glucose and plasma insulin response were significantly higher in the GLU+LEU treatment compared to the CON treatment. Plasma leucine concentrations increased after supplementation. During the post-exercise period isoleucine, valine and methionine concentrations decreased in both treatments but were significantly lower in the GLU+LEU treatment. There was no correlation between the insulin response and the response in plasma leucine, isoleucine, valine and methionine.

Conclusions

Repeated post-exercise administration with a mixture of leucine and glucose caused a marked insulin response and altered the plasma amino acid profile in horses in a similar manner as described in man. However, the decreases seen in plasma amino acids in horses seem to be related more to an effect of leucine and not to the insulin response as seen in man.     

亚洲棉bZIP蛋白家族的鉴定及GaFDs基因的组织表达分析

碱性亮氨酸拉链(basic leucine zipper, bZIP)是真核生物中数量最多并且最具多样性的转录因子之一,参与植物生长发育及响应生物和非生物胁迫。本研究利用亚洲棉(Gossypium arboreum)全基因组数据库,通过生物信息学分析分布在13条染色体上的159个bZIPs家族基因的全序列。系统进化、基因结构和保守基序分析表明这些基因分成13个亚家族。其中,A亚家族有3个GaFD基因GaFD1、GaFD2和GaFD3,通过实时荧光定量PCR分析3个GaFDs基因在不同组织中的表达,结果表明GaFD1和GaFD2在SAM中的表达量最高,GaFD3在茎中表达量最高。研究表明棉花基因组中具有数量众多的bZIP家族成员,不同基因结构及FD基因不同的表达特征表明bZIP基因在棉花生长发育中可能具有不同的功能,这些结果为进一步解析棉花bZIP家族基因的功能和作用机理积累了有价值的资料。     

饲粮添加亮氨酸和谷氨酸对肥育猪生长性能、胴体性状和肉品质的影响

本试验旨在探讨饲粮添加亮氨酸和谷氨酸对肥育猪生长性能、胴体性状和肉品质的影响。试验选取体重为77 kg左右的"杜×长×大"肥育猪60头,随机分为5个组,每组12头猪,公母各占1/2。对照组饲喂基础饲粮,试验组分别在基础饲粮中添加2.05%L-丙氨酸(等氮对照组)、1.00%亮氨酸+1.37%L-丙氨酸(亮氨酸组)、1.00%谷氨酸+1.44%L-丙氨酸(谷氨酸组)、1.00%亮氨酸+1.00%谷氨酸(亮氨酸+谷氨酸组)。试验期为60 d。结果表明:与对照组相比,谷氨酸组1~30 d的平均日采食量、末重和背膘厚均显著降低(P0.05),31~60 d的平均日增重降低了22.50%(P0.05);亮氨酸组、亮氨酸+谷氨酸组背最长肌和股二头肌中的肌内脂肪含量显著增加(P0.05),且亮氨酸+谷氨酸组1~30 d的平均日增重增加了8.04%(P0.05),31~60 d的平均日增重则降低23.70%(P0.05)。与等氮对照组相比,亮氨酸组和谷氨酸组的熟肉率、滴水损失和肉色均无显著差异(P0.05),亮氨酸+谷氨酸组肌肉黄度值显著降低(P0.05);各试验组背最长肌和股二头肌中风味氨基酸含量均无显著差异(P0.05)。上述结果提示,饲粮添加1.00%亮氨酸可降低1~30 d肥育猪的料重比,增加背最长肌肌内脂肪含量;饲粮添加1.00%谷氨酸或1.00%亮氨酸+1.00%谷氨酸可增加1~30 d的平均日增重,降低31~60 d的平均日增重;饲粮添加1.00%亮氨酸+1.00%谷氨酸可降低肉色黄度值,增加肌内脂肪含量,从而改善猪肉品质。     

日粮添加亮氨酸和苯丙氨酸对荷斯坦公犊生长性能及血清代谢物的影响

【目的】研究日粮添加亮氨酸和苯丙氨酸对犊牛生长性能和血清代谢物的影响,为亮氨酸和苯丙氨酸在奶牛高效生产中的应用提供理论参考。【方法】试验选取20头1日龄体重为(38±3)kg的荷斯坦公犊,随机分为4组,分别为对照组、亮氨酸组(1.435 g·L~(-1))、苯丙氨酸组(0.725 g·L~(-1))、混合氨基酸组(1.435 g·L~(-1)亮氨酸和0.725 g·L~(-1)苯丙氨酸),每组5头牛,各组以丙氨酸调节为等氮日粮。犊牛单独饲喂,试验共8周,预试期1周,正试期7周。试验犊牛单圈饲养,试验日粮由原奶和开食料组成。犊牛出生后,1 h内饲喂4.0 L初乳。此后1周为过渡期,日喂原奶2次,每次3.0 L。过渡期内以氨基酸添加量的20%为梯度逐天增加,至出生第6天达到添加量的100%。2—8周为正试期,日饲喂添加氨基酸的原奶2次。第2—3周每次3.5 L,4—8周每次4.0 L。第3周开始定量供给开食料,自由饮水。在犊牛1、3、5、7、8周龄测定体重、体高、体斜长和胸围,计算体躯指数和体长指数;采集10 mL血液用于获得血清,并检测其氨基酸组成、葡萄糖、尿素氮、胰岛素和胆囊收缩素。采用IBM SPSS Statistics V22.0统计软件GLM过程进行统计分析,采用LSD法进行多重比较。【结果】混合氨基酸组平均日增重显著低于其余3组(P0.05)。相对于对照组,各处理组显著提高最终体斜长(P0.05)。亮氨酸和苯丙氨酸具有降低最终体高的趋势(P0.10),而苯丙氨酸显著降低最终胸围(P0.05)。与对照组相比,亮氨酸组和混合氨基酸组显著提高犊牛8周龄体躯指数(P0.05)。但各组犊牛初始体重、体斜长、体高、胸围、体躯指数、体长指数和最终体重及体躯指数无显著差异(P0.05)。添加亮氨酸显著提高血清组氨酸、亮氨酸、苏氨酸、甘氨酸和总必需氨基酸的浓度(P0.05),趋于增加天冬氨酸和总氨酸浓度(P0.10),但显著降低丙氨酸和甘氨酸浓度(P0.05)。苯丙氨酸显著提高血清苯丙氨酸、谷氨酸和甘氨酸浓度(P0.05),对照组因添加丙氨酸调节为等氮日粮,其血清丙氨酸浓度显著高于亮氨酸组与混合氨基酸组(P0.05)。苯丙氨酸和亮氨酸对血清各种氨基酸浓度无交互效应(P0.05)。亮氨酸显著提高血清葡萄糖浓度(P0.05),苯丙氨酸降低血清葡萄糖浓度(P0.05),且与亮氨酸存在负交互效应(P0.05)。此外,苯丙氨酸显著提高血清尿素氮含量(P0.05),但亮氨酸和苯丙氨酸添加对血清中胰岛素及胆囊收缩素浓度无显著影响(P0.05)。【结论】日粮中添加亮氨酸可提高犊牛血清部分必需氨基酸、葡萄糖浓度、8周龄体躯指数和平均日增重。从以上指标可见,亮氨酸添加效果优于苯丙氨酸,但二者在影响血清葡萄糖浓度方面存在负交互效应。     

大豆GmHAT5的克隆及其转基因百脉根的抗盐分析

【目的】从大豆盐胁迫基因表达谱中筛选并克隆得到同源异型域亮氨酸拉链蛋白(HD-Zip)家族基因GmHAT5,将其转化豆科模式植物百脉根并进一步探究其抗盐调控机制。【方法】使用多种生物信息学软件对GmHAT5的开放读码框(ORF)、编码蛋白的分子量、等电点、序列结构和蛋白定位等进行预测,同时将大豆GmHAT5蛋白与其他10个物种的同源蛋白进行比对分析,并对GmHAT5在大豆不同器官及盐胁迫下的表达特性进行分析。此外,构建GmHAT5的植物超表达载体,通过对发根农杆菌LBA1334的转化,得到"复合体"百脉根植株,并在盐胁迫条件下对其进行抗盐表型分析;通过对根癌农杆菌EHA105的转化,获得百脉根的稳定转化株系,并对其进行抗盐表型分析及相关生理指标检测。【结果】多种生物信息学软件分析表明,该片段包含1个1 038 bp的ORF,编码345个氨基酸。GmHAT5的理论分子量和等电点分别为39.17 k D和4.63。GmHAT5蛋白定位于细胞核,与其他HD-Zip家族蛋白一样,属于典型的核蛋白。序列分析表明,GmHAT5包含一个同源异型结构域和一个亮氨酸拉链结构域,属于第I类同源异型域亮氨酸拉链蛋白。同源蛋白比对表明其与野生大豆GsHAT5同源性最高。基因表达特性分析表明,GmHAT5在大豆植株的各个不同器官中均有表达,是一个受盐诱导上调表达的HD-Zip类转录因子。发状根转化的结果表明,使用200 mmol·L~(-1) NaCl处理植株7 d后,"复合体"植株生长状态良好,对照组植株叶片明显萎蔫、失绿;不同NaCl浓度处理离体转基因发状根14 d后,对照组较试验组发状根明显干枯、生长受到抑制。稳定转化结果显示,不同盐浓度处理14 d后,转GmHAT5百脉根与两组对照植株相比生长状态更好。相关生理指标检测结果显示,与两组对照相比,转基因百脉根植株中丙二醛含量和相对质膜透性明显降低(P0.05),而叶绿素含量和根系活力则显著升高(P0.05)。测定不同植株阳离子含量的结果表明,转基因百脉根株系与两组对照相比,Na~+在叶片和根中含量显著下降,K~+和Ca~(2+)在叶片和根中含量显著升高。【结论】从大豆中克隆得到一个编码HD-ZipⅠ类同源异型域亮氨酸拉链蛋白基因GmHAT5,该基因在大豆中受盐胁迫诱导表达量显著升高。超量表达GmHAT5显著增强百脉根的耐盐能力,发状根转化法可以作为一种快速有效筛选抗盐候选基因的手段。推测GmHAT5在大豆盐胁迫应激调控中扮演重要角色。