Using RT-PCR and glutathione level to study the effect of follicular fluid on in vitro maturation and gene expression of sheep oocytes

The aim of this study is to evaluate the effect of sheep follicular fluid (SFF) supplementation of the in vitro maturation (IVM) media of sheep oocytes on the resumption of meiosis, glutathione (GSH) level, and expression of apoptosis (Bax, Bcl-2) as well as heat shock protein beta-1 (HSPB1) genes. Sheep ovaries were collected from the central slaughterhouse of Riyadh city, KSA. Oocytes were aspirated from 3 to 8 mm follicles. Sheep oocytes were cultured in maturation medium with different concentrations of sheep follicular fluid: 0% (control), 10%, 20% and 40% for 24 h. The results indicated that the maturation rate of oocytes was significantly (p ≤ .05) decreased in 40% SFF (36.87%) versus the control (61.3%), 10% SFF (63.95%) and 20% SFF (64.08%). The supplementation of the IVM medium with 10% SFF induced an intra-oocyte GSH concentration that was significantly higher than in sheep oocytes cultured with 20% and 40% SFF and similar to the GSH content in oocytes cultured without SFF. Real-time polymerase chain reaction analysis of gene expression revealed no significant differences in the Bax and HSPB1 genes between the control and 10% SFF, whereas they were significantly higher in 40% FF (p ≤ .05) compared to the control. The expression of Bax:Bcl-2 was significantly higher in 20% and 40% SFF compared to the control group. In conclusion, the addition of SFF to the IVM culture of sheep oocytes is recommended to support nuclear maturation and increase oocyte competence.     

An unstable gene controlling developmental variegation in okra (Abelmoschus esculentus (L.) Moench)

Summary Genetic studies on radiation-induced chlorina and variegated mutants of okra (Abelmoschus esculentus (L.) Moench) revealed the existence of an unstable gene. The normal green color of the leaves is controlled by duplicate genes C₁ and C₂, either of which produces the green colour. The chlorina plants are C ₁ C ₁ C ₂ C ₂. The allele c ₁ ^v is dominant to both C ₁ and C ₂ but is unstable. The homozygote c ₁ ^v c ₁ ^v c ₂ c ₂ is a normal green while the heterozygote c _i ^v c ₁ c ₂ c ₂ has a variegated phenotype as a result of the mutation of c ₁ ^v to c ₁ during development. In green plants with a c ₁ ^v c{sh1/v}c ₂ c ₂ genotype, the autonomous mutation of one of the c ₁ ^v alleles to c ₁ may take place at the pre-meiotic stage. In the variegated genotype (c ₁ ^v c ₁ c ₂ c ₂), the mutation of c ₁ to c ₁ ^v may take place in early ontogeny, thus producing green plants. The allele C ₁, when associated with c ₁ ^v in a heterozygous condition, mutates to c ₁ at the pre-meiotic stage even in the presence of the allele C ₂.     

苹果柠檬酸合酶3基因家族成员鉴定及表达分析

柠檬酸合酶(citrate synthase 3, CS3)是细胞代谢途径中的关键酶之一,其活性调节着生物体的物质和能量代谢过程。本研究旨在从苹果全基因组中鉴定CS3基因家族成员,并进行生物信息学和表达模式分析,为研究苹果CS3基因的潜在功能提供理论基础。利用BLASTp基于GDR数据库鉴定苹果CS3家族成员,通过Pfam、SMART、MEGA5.0、clustalx.exe、ExPASy Proteomics Server、MEGAX、SOPMA、MEME和WoLF PSORT等软件分析CS3蛋白序列基本信息、亚细胞定位情况、结构域组成、系统进化关系以及染色体定位情况。利用酸含量的测定和实时荧光定量PCR (real-time fluorescence quantitative polymerase chain reaction, qRT-PCR)技术检测苹果6个CS3的组织表达和诱导表达特性。苹果CS3基因家族包含6个成员,这些CS3蛋白包括473−608个不等的氨基酸残基,等电点分布在7.21−8.82。亚细胞定位结果显示CS3蛋白分别定位在线粒体和叶绿体。系统进化分析可将其分为3类,各亚家族基因数量分别为2个。染色体定位结果显示,CS3基因分布在苹果不同的染色体上。蛋白二级结构以a-螺旋为主,其次是无规则卷曲,b-转角所占比例最小。筛选的6个家族成员在不同苹果组织中均有表达,整体表达趋势从高到低依次为MdCS3.4相对表达含量最高,MdCS3.6次之,其他家族成员相对表达量依次为MdCS3.3>MdCS3.2>MdCS3.1>MdCS3.5。qRT-PCR结果显示,MdCS3.1和MdCS3.3基因在酸含量较低的‘成纪1号’果肉中相对表达量最高,酸含量较高的‘艾斯达’果肉中MdCS3.2和MdCS3.3基因相对表达量最高。因此,本研究对不同苹果品种中CS3基因相对表达量进行了检测,并分析了其在苹果果实酸合成过程中的作用。结果表明,CS3基因在不同苹果品种中的相对表达量存在差异,为后续研究苹果品质形成机制提供了参考。     

核、质基因对肺形侧耳菌丝生长、营养成分及基因表达的影响

本研究通过杂交构建肺形侧耳同核异质菌株N1M1、N1M2和同质异核菌株N1M1、N2M1,比较菌丝形态与生长速度、营养成分以及常见的细胞核基因与线粒体基因的表达量,分析线粒体基因对肺形侧耳菌丝的影响,探讨线粒体基因与核基因的相互作用。由菌丝生长情况可知N1M1和N1M2菌丝形态相似,生长速度差异不显著,N1M1和N2M1菌丝形态差异大,生长速度差异极显著,在菌丝形态与生长速度上细胞核基因作用大于线粒体基因。进一步检测菌株中的主要营养成分发现必需氨基酸与总水解氨基酸含量差异显著,菌株N1M2蛋白含量显著高于N1M1,N1M1维生素C含量是N1M2的1.67倍,菌株N2M1多糖和蛋白含量显著高于N1M1,铁和维生素C含量显著低于N1M1。所以细胞核基因、线粒体基因都能影响肺形侧耳营养成分含量。检测同核异质菌株N1M1、N1M2的7个细胞核常见基因的表达情况发现,N1M2菌丝中6个细胞核基因的表达量都显著高于N1M1,这表明肺形侧耳线粒体基因的不同会影响核基因的表达;同质异核菌株N1M1、N2M1的14个线粒体普通编码蛋白基因表达差异显著,这说明线粒体基因的表达量会因核基因的不同有所差异。综上,肺形侧耳线粒体基因和细胞核基因能够相互影响,共同作用于生命活动。     

不同树龄水曲柳半同胞家系生长性状变异研究

为筛选优质的水曲柳种质资源,以吉林省三岔子林业局国家水曲柳良种基地的16年生水曲柳子代测定林75个半同胞家系为材料,分别在第4、11和16年对其树高、地径（胸径）和材积进行测定。方差分析结果表明,不同变异来源各指标均达极显著差异水平（P<0.01）;各指标表型变异系数变化范围为22.20%~63.30%,家系遗传力变化范围为0.796~0.981;相关性分析结果表明各指标间均呈极显著正相关;以材积为评价指标,按10%入选率对家系进行评价选择,初步筛选8个优良家系,入选的家系材积平均值比总平均值高0.007 m³,遗传增益为36.03%;该研究选出的优良家系可为良种登记提供基础,也可为种子园的改建提供材料。     

遗传性骨病致病基因及相关miRNA的相互作用分析

目的:筛选遗传性骨病相关的致病基因和其相关的mi RNA,并研究两者相互作用关系以及在遗传性骨病中的作用。方法:本研究应用生物信息学的方法,首先应用mirwalk和《国际遗传性骨病分类标准》分析遗传性骨病的致病基因,根据筛选出来的致病基因利用mirwalk的10个预测mirna搜索引擎功能,搜索致病基因的相关mi RNA,并应用excel工作表统计分析mirna的靶向致病基因;再应用Cytoscape软件分析致病基因和相关mirna之间的相互作用关系。结果:本研究中与遗传性骨病密切相关的基因可以分为四类:第一类为成骨不全类基因COL1A1、COL1A2等;第二类为骨密度降低类基因如ACVR1、ALX1等;第三类为骨密度增加类基因如CASR、DMTF1等;第四类为通过作用骨干参与骨密度增加的基因如TGFBR1、MYCN等。其中与成骨不全关系最为密切的mirna是hsa-miR-26b、hsa-miR-19、hsa-miR-200c;与骨密度降低关系最为密切的mirna是hsa-miR-138、hsa-miR-505;与骨密度增加关系最为密切的mirna是hsa-miR-196a、hsa-miR-200b、hsa-miR-19b。结论:mirna可通过调控遗传性骨病致病基因在其病理过程中起到重要作用,提示上述mirna可能是成为遗传性骨病产前筛查和临床药物治疗的新靶点。