不同启动子调控的DREB1A基因对黄瓜的遗传转化

以黄瓜(CucumissativusL.)主栽品种长春密刺的子叶节为受体材料,采用农杆菌介导法,将分别由诱导型启动子rd29A和组成型启动子35S调控的转录因子DREB1A基因导入黄瓜,获得大量PCR阳性植株。统计结果表明:诱导型启动子rd29A调控的DREB1A基因抗性芽率和再生植株PCR阳性率明显高于组成型启动子35S。     

猪心肌和骨骼肌miRNA转录组比较分析

【目的】研究microRNA(miRNA)在猪不同类型肌肉生长发育中的作用。【方法】以猪心肌以及2种骨骼肌(背最长肌和腰大肌)为材料,采用Illumina高通量测序技术鉴定其miRNA转录组,并进行差异表达分析和靶基因富集分析。【结果】从猪心肌、背最长肌和腰大肌组织的3个小RNA测序文库中共获得约56M的序列,通过生物信息分析共鉴定出907个非冗余miRNA,其中441个miRNA在3个文库共同表达,123个miRNA在文库间表现为显著的差异表达(P10-6)。对3个文库差异表达miRNA的相关性分析发现,背最长肌与腰大肌的相关性最高(r=0.94),证明2种骨骼肌的表达模式十分相近,而心肌与2种骨骼肌的表达模式不同。通过对各文库高表达的miRNA分析发现,表达量前十的miRNA中有6个miRNA的表达量在心肌中极显著上调。对在心肌内上调的6个miRNA进行的靶基因预测和生物学功能预测发现,这些差异miRNA主要靶向作用于钙信号通路和胰岛素信号通路。荧光定量PCR(qRT-PCR)验证结果表明高通量测序结果准确可靠。【结论】从miRNA层面揭示了猪心肌和骨骼肌的差异,这种差异在一定程度上印证了心肌和骨骼肌之间迥异的生理学特征。     

高邮鸭IGF-1基因SNP位点变异与骨骼肌生长发育相关性分析

胰岛素样生长因子1(IGF-1)是动物生长轴重要基因之一,在调节动物生长发育和代谢中具有重要作用。采用LDR法检测IGF-1基因在高邮鸭群体中多态性,筛选与高邮鸭骨骼肌生长性状相关SNP位点。结果表明,在IGF-1基因第二内含子上检测到g.110 431GT突变,形成GG、GT、TT三种基因型,该突变位点具有中度多态信息含量,在高邮鸭中处于哈代-温伯非平衡状态。相关分析表明,该突变位点与70日龄体重、胸腿肌重、胸肌肌纤维面积和直径、腿肌肌纤维密度显著相关,可作为高邮鸭生长发育早期选育分子标记。     

SFRP2 affects prenatal muscle development and is regulated by micro RNA-1/206 in pigs

Secreted frizzled-related protein 2(SFRP2), a member of the SFRPs family, is associated with cell growth and differentiation in myogenesis. Our previous study suggested that SFRP2 was a potential target of micro RNA(mi RNA)-1/206, which was considered as myomiR s. To further explore the biological function and regulation mechanisms of the SFRP2 gene in porcine skeletal muscle development, we first analyzed the sequence structure of the porcine SFRP2 gene. Subsequently, we detected its tissue distribution in adult Tongcheng pigs(a Chinese indigenous breed) and investigated its dynamic expression in developmental skeletal muscle(13 prenatal and 7 postnatal time points) in Tongcheng pigs. An interaction analysis between SFRP2 and myomi Rs was also performed. The results showed that the expression pattern of the SFRP2 varied greatly across diverse tissues. It exhibited abundant expression in prenatal skeletal muscle and peaked at 55 days post coitus(E55), and had a lower expression in postnatal skeletal muscle, indicating that the SFRP2 gene might affect porcine embryonic skeletal muscle development. Co-expression analysis revealed that the expression levels of SFRP2 correlated negatively with mi RNA-1(r=–0.570, P-value=0.009) and mi RNA-206(r=–0.546, P-value=0.013), but positively with SFRP1(r=0.613, P-value=0.004). The bioinformatics analysis and dual luciferase assay verified that the SFRP2 was a putative target of mi RNA-1/206 in pigs. Therefore, this study is helpful for understanding the biological function and molecular regulation of the SFRP2 gene during porcine skeletal muscle development.     

外源T3对鸭胚骨骼肌发育和MyoD mRNA表达的影响

甲状腺激素(thyroid hormones,THs)是体内肌肉发育的重要调节因子,MyoD在骨骼肌特异基因的转录调控,肌细胞的定向和分化中起着主要作用。为探讨三碘甲状腺原氨酸(triiodothyronine,T3)对鸭胚骨骼肌生长发育和Myod mRNA表达的影响,分别注射100μL含0.25μg(注射组)和0μg(对照组)T3的生理盐水溶液到入孵前的鸭种蛋蛋清内,检测鸭胚骨骼肌发育和MyoD mRNA的表达情况。结果表明,外源T3对鸭胚胸肌率、腿肌重和腿肌率的影响不显著(P0.05),但显著提高27胚龄鸭胚的体重和胸肌重(P0.05);胸肌MyoD mRNA表达与胸肌重的变化趋势相一致,其中27胚龄注射组胸肌的表达水平显著高于对照组(P0.05);注射组腿肌MyoD mRNA表达在13胚龄显著提高(P0.05),与腿肌发育早于胸肌相一致;注射组胸肌MyoD mRNA表达与胸肌重呈显著正线性相关,胸肌率呈负相关,腿肌MyoD mRNA与腿肌重呈负线性相关。结果显示,外源T3提高了出雏前鸭胚的体重和胸肌重,可能是通过调节MyoD mRNA表达参与胸肌的生长发育。     

松墨天牛肌钙蛋白T基因全长cDNA克隆及分析

采用c DNA末端快速扩增(RACE)方法获得了松墨天牛(Monochamus alternatus)肌钙蛋白T基因c DNA,全长1531bp,命名为Ma Tn T(Gen Bank:KJ756400).该基因开放阅读框(ORF)为1020 bp,编码339个氨基酸,等电点为9.26,推测的编码蛋白质分子质量为37.29 ku.同源比对结果表明:Ma Tn T与褐飞虱(Nilaparvata lugens)、黑腹果蝇(Drosophila melanogaster)等12种昆虫肌钙蛋白氨基酸同源性为87%-88%;构建的系统发育树显示Ma Tn T处于独立分支;Ma Tn T没有信号肽和跨膜螺旋,属于亲水性氨基酸,二级结构包含了螺旋和不规则卷曲两种形式,含有丝氨酸(Ser)、苏氨酸(Thr)、酪氨酸(Tyr)磷酸化位点数分别为8、9、3个.RT-q PCR检测表明,Ma Tn T基因在幼虫、蛹和成虫中均有表达;在成虫触角和足中表达量最高.     

Regulation of mitochondrial biogenesis in skeletal muscle by CaMK

Endurance exercise training promotes mitochondrial biogenesis in skeletal muscle and enhances muscle oxidative capacity, but the signaling mechanisms involved are poorly understood. To investigate this adaptive process, we generated transgenic mice that selectively express in skeletal muscle a constitutively active form of calcium/calmodulin-dependent protein kinase IV (CaMKIV*). Skeletal muscles from these mice showed augmented mitochondrial DNA replication and mitochondrial biogenesis, up-regulation of mitochondrial enzymes involved in fatty acid metabolism and electron transport, and reduced susceptibility to fatigue during repetitive contractions. CaMK induced expression of peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1), a master regulator of mitochondrial biogenesis in vivo, and activated the PGC-1 gene promoter in cultured myocytes. Thus, a calcium-regulated signaling pathway controls mitochondrial biogenesis in mammalian cells.     

Effect of membrane potential changes on the calcium transient in single rat cardiac muscle cells

The mechanism that links membrane potential changes to the release of calcium from internal stores to cause contraction of cardiac cells is unclear. By using the calcium indicator fura-2 under voltage-clamp conditions, changes in intracellular calcium could be monitored in single rat ventricular cells while controlling membrane potential. The voltage dependence of the depolarization-induced increase in intracellular calcium was not the same as that of the calcium current (Isi), which suggests that only a small fraction of Isi is required to trigger calcium release from the sarcoplasmic reticulum. In addition, sarcoplasmic reticulum calcium release may be partly regulated by membrane potential, since repolarization could terminate the rise in intracellular calcium. Thus, changes in the action potential will have immediate effects on the time course of the calcium transient beyond those associated with its effects on Isi.     

Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region

We surveyed an Anopheles gambiae population in a West African malaria transmission zone for naturally occurring genetic loci that control mosquito infection with the human malaria parasite, Plasmodium falciparum. The strongest Plasmodium resistance loci cluster in a small region of chromosome 2L and each locus explains at least 89% of parasite-free mosquitoes in independent pedigrees. Together, the clustered loci form a genomic Plasmodium-resistance island that explains most of the genetic variation for malaria parasite infection of mosquitoes in nature. Among the candidate genes in this chromosome region, RNA interference knockdown assays confirm a role in Plasmodium resistance for Anopheles Plasmodium-responsive leucine-rich repeat 1 (APL1), encoding a leucine-rich repeat protein that is similar to molecules involved in natural pathogen resistance mechanisms in plants and mammals.     

DNA synthesis in differentiating skeletal muscle cells: initiation by ultraviolet light

As skeletal muscle cells differentiate, they fail to initiate DNA synthesis. This rigid regulation, which persists even after cells are fully developed, does not extend to "repair" DNA synthesis, in that ultraviolet light initiates DNA synthesis in 99 percent of the muscle nuclei exposed. The rate of "repair" DNA synthesis in these nuclei, however, drops over 50 percent at the time of cell differentiation.     

不同品种鸭胚胎期骨骼肌成肌细胞GHR和IGF 1 mRNA表达差异分析

为探讨生长激素受体（growth hormone receptor,GHR）和胰岛素样生长因子 1（Insulin like growth factor 1,IGF 1）在不同品种鸭胚胎期骨骼肌成肌细胞中的表达差异。选择生长速度不同的金定鸭和高邮鸭为试验动物,采用实时荧光定量PCR方法检测鸭13,15,17,19,21和23胚龄时胸肌和腿肌成肌细胞GHR,IGF 1 mRNA的表达情况,并进行了品种间比较。结果发现：13胚龄时,高邮鸭胸肌和腿肌成肌细胞IGF 1的表达均显著高于金定鸭（P<0.05）;17胚龄是两个品种鸭GHR和IGF 1共同的表达高峰期;17胚龄之后,鸭胸肌和腿肌成肌细胞GHR和IGF 1表达均显著降低。在19,21胚龄时,高邮鸭胸肌成肌细胞2个基因的表达均显著高于金定鸭（P<0.05）,而腿肌成肌细胞GHR和IGF 1表达在品种间无显著差异（P>0.05）。鸭胚胸肌和腿肌成肌细胞GHR和IGF 1表达呈现极显著的正线性相关（P<0.01）。提示：GHR和IGF 1的表达谱及各自在品种间的变化规律基本一致;鸭胚骨骼肌成肌细胞GHR和IGF 1 mRNA表达有组织特异性,二者之间可能存在正调控的作用机制。     

Phosphate release and force generation in skeletal muscle fibers

Rapid laser pulse-induced photolysis of an adenosine triphosphate precursor in muscle fibers abruptly initiated cycling of the cross-bridges. The accompanying changes in tension and stiffness were related to elementary mechanochemical events of the energy-transducing mechanism. When inorganic phosphate was present at millimolar concentrations during liberation of adenosine triphosphate in the absence of calcium, relaxation was accelerated. Steady active tension in the presence of calcium was decreased but the approach to final tension was more rapid. These results suggest that, during energy transduction, formation of the dominant force-generating cross-bridge state is coupled to release of inorganic phosphate in a reaction that is readily reversible.     

不同形态氮素调控番茄果实和叶片内有机酸代谢的研究进展

硝态氮(NO3--N)和铵态氮(NH4+-N)是植物吸收的两种主要氮素形态.由于植物对NO3-和NH4+吸收和同化的机制不同,导致叶片及果实中有机酸含量差异很大.本文阐述了不同形态氮素对植物叶片内有机酸合成的影响,指出供NO3-植物叶片中苹果酸、α-酮戊二酸及柠檬酸含量均显著高于供NH4+植物.番茄果实中苹果酸和柠檬酸是两种含量最多的有机酸,也是影响果实品质的重要因素;进一步分析了番茄果实发育过程中有机酸种类、含量及相关有机酸代谢酶活性的变化,探讨了不同形态氮素对番茄果实中苹果酸和柠檬酸含量以及氮代谢和有机酸代谢关键酶活性的影响.提出氮素形态可能对果实中有机酸含量有重要的调控作用,以期为农业生产中采用不同形态氮素调控果实有机酸代谢提供理论依据.     

蛋内注射T3对鸭胚骨骼肌发育和MSTN mRNA表达的影响

为探讨生长因子三碘甲状腺原氨酸(Triiodothyronine,T3)对鸭胚骨骼肌生长发育和肌肉生长抑制素(Myostatin,MSTN)mRNA表达的影响,在入孵前的鸭种蛋蛋清内分别注射100μl的生理盐水溶液(对照组)和含0.25μg T3的生理盐水溶液(注射组),检测13、17、21、25、27胚龄鸭胸肌和腿肌发育及骨骼肌MSTN mRNA的表达情况。结果显示,T3处理极显著提高27胚龄鸭胚的胸肌质量,但对腿肌质量影响不显著;21胚龄注射组胸肌MSTN mRNA表达显著高于对照组,27胚龄则显著低于对照组;注射组腿肌MSTN mRNA表达仅在25胚龄显著增加。相关性分析结果显示,两组骨骼肌质量与MSTN mRNA表达呈不同程度的线性相关。表明蛋内注射T3能增加鸭胚胸肌质量,并改变MSTN mRNA表达量,MSTN mRNA的差异表达可能在鸭胚胸肌发育过程中具有重要的作用。     

A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle

A high proportion of purebred Hampshire pigs carries the dominant RN- mutation, which causes high glycogen content in skeletal muscle. The mutation has beneficial effects on meat content but detrimental effects on processing yield. Here, it is shown that the mutation is a nonconservative substitution (R200Q) in the PRKAG3 gene, which encodes a muscle-specific isoform of the regulatory gamma subunit of adenosine monophosphate-activated protein kinase (AMPK). Loss-of-function mutations in the homologous gene in yeast (SNF4) cause defects in glucose metabolism, including glycogen storage. Further analysis of the PRKAG3 signaling pathway may provide insights into muscle physiology as well as the pathogenesis of noninsulin-dependent diabetes mellitus in humans, a metabolic disorder associated with impaired glycogen synthesis.     

rd29A和CaMV-35S启动子调控转AtDREB2A苜蓿耐碱性分析

rd29A和CaMV-35S启动子广泛应用于植物基因工程中,但调控效果在不同转基因植物中不同。文章采用Real-time PCR分析转基因各株系中AtDREB2A基因表达差异;对苜蓿扦插苗及一年生转基因苜蓿成苗分别作50 mmol·L~(-1)NaHCO_3(pH 8.0)和混合盐碱土(pH 9.3)处理,统计苜蓿各株系成活率、开花植株数,测定叶绿素、丙二醛、相对电导率及根系活力。结果表明,两种启动子对AtDREB2A表达的调控存在明显差异,35S启动子调控的AtDREB2A为超量表达,碱胁迫处理后显著上调,达57.6倍;rd29A启动子调控AtDREB2A诱导表达,表达量低于35S启动子调控株系(20.7倍),AtDREB2A超量表达抑制植株正常生长。在幼苗期和成苗期,两种启动子各转基因株系均有一定耐碱能力,但存在差异。AtDREB2A诱导表达耐碱性效果更明显,其叶绿素含量、相对电导率、MDA、根系活力变化均显著低于AtDREB2A超量表达。研究两种启动子调控的转AtDREB2A基因苜蓿耐碱效果,为AtDREB2A基因在苜蓿耐碱基因工程中应用提供方法。