中国地方绵羊群体TYRP1基因遗传变异研究

本研究旨在了解酪氨酸酶相关蛋白1（tyrosinase related protein 1，TYRP1）基因在中国地方绵羊群体内的遗传变异，以及TYRP1基因突变与不同毛色表型绵羊群体的相关性。通过直接测序法和PCR-RFLP技术对10个中国地方绵羊群体进行单核苷酸多态性（SNP）检测，利用Beagle、PLINK和POPGENE等软件对突变位点数据进行单倍型构建、连锁不平衡分析和遗传变异研究。突变位点检测结果表明，在绵羊TYRP1基因内识别了13个SNPs，其中位于TYRP1基因外显子上的10个SNPs位点，除个别位点在大尾寒羊、中国美利奴羊和岷县黑裘皮羊中没有发生突变外，其他突变位点在所有绵羊品种中均出现不同程度变异，说明中国地方绵羊群体具有较高的遗传多样性。单倍型分析结果表明，所有样本中共有42个单倍型，优势单倍型0000000000（245/918）、0100000001（91/918）在所有绵羊群体中均存在，除单倍型0101100000（93/918）在中国美利奴羊中没有出现，单倍型0001000001（69/918）在岷县黑裘皮羊、哈萨克羊群体中没有出现外，在其他群体中均存在。连锁分析结果表明，10个SNPs在所有样本中均存在2个连锁模块。群体遗传变异分析表明，中国地方绵羊群体具有较高水平的群体内遗传变异，各绵羊品种间存在明显的遗传分化模式，且各品种遗传关系与其品种传统分类结果基本一致。本研究为进一步研究TYRP1基因对绵羊毛色遗传性状的影响提供了参考依据。     

不同因素影响下层状土壤水分入渗特征及水力学参数估计

层状土壤是自然界常见的土体结构,其水分运移规律不同于均质土;大气降水、灌溉水等水分的入渗是土壤水文过程的一个重要环节,同时它也与地下水补给、污染物运移等过程紧密相关。土壤初始含水量、土体构型及供水强度等因素均会影响水分的入渗过程。为探究积水深度、土体构型、初始含水量三种因素对层状土壤水分运移的影响,通过室内积水入渗试验对湿润锋、累积入渗量、土壤剖面压力水头进行观测,并利用Hydrus-1D模型反演水力参数并对相应条件下的水分运移规律进行模拟和分析。结果表明,层状土壤中湿润锋随时间的推进方式由非线性过渡至线性,入渗率逐渐减小。三种因素作用下,层状土壤水分运移特征有明显差异:积水深度、土壤初始含水量增加时,湿润锋运移速率和入渗率均增大,且各观测点压力水头升高加快,土壤不饱和程度降低;上砂壤下粉砂壤构型较上粉砂壤下砂壤构型而言,整体湿润锋推进速率和入渗率较大,出流快,且入渗后期界面处的压力水头高于其他观测点。且结果表明,反演的水力学参数较拟合实测的参数更适用于层状土壤入渗的模拟和预测。该研究旨在揭示和掌握层状土壤水分运移规律和影响因素的作用机制,并进一步为农田灌溉措施的合理制定提供科学依据。     

利用CRISPR/Cas9技术编辑BADH2-1/BADH2-2创制香米味道玉米新种质

【目的】香味是作物的重要食味品质之一。2-乙酰-1-吡咯啉（2-acetyl-1-pyrroline,2-AP）是主要香味物。BADH2是控制水稻等作物香味性状的关键基因,敲除该基因可以产生香味稻米。利用CRISPR/Cas9基因编辑技术在北京市农林科学院自育的玉米骨干亲本京724上敲除BADH2同源基因,以期获得有香米味道的玉米新种质材料。【方法】利用Ensembl数据库在线BLAST工具,将水稻OsBADH2蛋白序列在拟南芥、水稻和玉米蛋白序列数据库中进行序列比对,获得上述3个物种的BADH基因家族成员,并利用UniProt蛋白数据库中的结构域信息进行验证。进一步使用MEGA软件进行系统进化分析,获得玉米BADH2同源基因作为候选编辑靶标。基于CRISPR/Cas9基因编辑技术的原理,在候选基因的外显子处设计特异性靶点,并构建入CRISPR/Cas9基因编辑载体。再以玉米自交系京724为受体,利用农杆菌介导的遗传转化方法,通过磷酸甘露糖异构酶基因（phosphomannose isomerase,PMI）抗性筛选获得阳性转基因植株。转基因株系经测序明确其在靶基因中产生的突变类型。利用气相色谱质谱联用仪（gas chromatography-mass spectrometry,GC-MS）检测基因编辑株系T₁籽粒中香米主要香味物质2-AP的含量,以确认京724在基因编辑前后2-AP含量的变化。【结果】系统进化分析发现,玉米中存在2个BADH2同源基因,分别命名为ZmBADH2-1和ZmBADH2-2。ZmBADH2-1位于第4染色体,ZmBADH2-2位于第1染色体。2个基因均包含15个外显子和14个内含子,第4外显子间的核苷酸序列高度同源。在2个基因的第4外显子区域设计靶点并构建入CRISPR/Cas9基因编辑载体,通过遗传转化获得28株转基因株系。PCR扩增及测序分析结果显示,其中10株材料的2个ZmBADH2s在靶点区域均发生突变,突变基因型包括双等位突变和多等位突变,突变类型为不同数量的碱基缺失和插入。质谱检测结果显示玉米ZmBADH2双基因突变体籽粒中存在与香稻同样成分的2-AP。随机选取的4个T₁代基因编辑株系籽粒中,2-AP平均含量分别为438.29、404.63、348.65和161.82 μg·kg^-1,而未经过编辑的京724中未检测到2-AP。【结论】利用CRISPR/Cas9技术对玉米ZmBADH2-1和ZmBADH2-2同时进行定点敲除,创制出籽粒中具有香米味道的玉米骨干亲本新种质材料。     

An improved method for extraction of sorghum polymeric protein complexes

A method for fractionating sorghum proteins using extraction solvents and techniques designed to obtain polymeric protein structures (especially disulfide linked) was developed. Extraction and separation conditions were optimized in terms of completeness of protein extraction, sample stability, and analytical resolution. After pre-extraction of albumins and globulins, a 3-step sequential procedure involving no reducing agents was applied to ground whole sorghum flour. The three fractions obtained represented proportionally different protein polymer contents and molecular weight distribution as evidenced by comparative size exclusion chromatography. Protein composition also varied among the extracts with differences in kafirin composition and non-kafirin proteins detected in the fractions by RP-HPLC and SDS-PAGE analysis. The ability to quantify and further characterize sorghum polymeric protein complexes will be useful for additional studies linking protein structures with functionality and digestibility and variations for these properties within diverse sorghum germplasm.     

New angiotensin-converting enzyme inhibitory peptide from Coix prolamin and its influence on the gene expression of renin-angiotensin system in vein endothelial cells

Coix seed, which is a traditional Chinese medicine, has been used to treat hypertension for thousands of years. It has been shown that Coix prolamin peptides display high levels of angiotensin I converting enzyme (ACE) inhibitory activity. Hence, we purified the ACE inhibitory peptides from Coix prolamin hydrolysates and evaluated the influence of the most potent peptide on the renin-angiotensin system (RAS) genes expression in human umbilical vein endothelial cells (HUVECs). In this study, Coix prolamin peptides were sequentially separated by ultrafiltration, ion exchange chromatography, gel filtration chromatography and RP-HPLC, while the peptide structure was analyzed by mass spectrometry. Next, in silico proteolysis, pharmacophore and molecular docking were further applied to screen and optimize the structure of peptides. Finally, a novel ACE inhibitory peptide VDMF was obtained, in which its influence on the gene expression of RAS signaling pathway in AngⅡ-injury HUVECs was evaluated by quantitative real-time PCR. VDMF significantly down-regulated ACE, AngII type 1 receptor (AT1R) and ACE2 mRNA expression in comparation with model group, while up-regulating Mas gene expression. Hence, we obtained a novel antihypertensive candidate that was derived from the Coix peptides, which could involve a multi-modulation mechanism that regulates blood pressure.     

Characterization and bioactivities of young rice protein hydrolysates

Immature rice was reported to contain higher quantities of bioactive compounds than mature rice. Young rice protein is easy to digest and has hypoallergenic potential, with protein content of 7.2–11.5% compared to rice bran at 9.8%. Few studies have reported on bioactivities and characterization of young rice proteins and their hydrolysates. Bioactivities of native protein and protein hydrolysates of two rice varieties (white rice and colored rice) were characterized and investigated for four development stages (flowery, milky, dough, and mature). Degree of hydrolysis of young rice protein was considerably higher than at the mature stage. Highest DPPH and iron chelating activity were found in alcalase® protein hydrolysate during the flowery-to-milky stage. Iron chelating activity was constant in all development stages because of the low polar amino acid content in rice. The ACE activity of alcalase® protein hydrolysate was higher than native protein at the same development stage, as observed in the milky and dough stages. Inhibitory activity of young rice hydrolysate HepG2 cells was concentration-dependent and not correlated with protein molecular size.     

外源氯化钙对低温胁迫下胡椒抗寒生理指标的影响

以中国胡椒主栽品种‘热引1号’胡椒为材料,设置4个不同浓度氯化钙（7、14、21、28 mmol/L）处理,以去离子水为对照,低温胁迫4 d（10 ℃/5 ℃,12 h/12 h）,恢复培养6 d（28 ℃/20 ℃,12 h/12 h）,观察表型并测定各处理的光合参数、抗氧化酶活性及渗透性物质含量变化情况。结果表明：7 mmol/L氯化钙处理胡椒可以改善寒害表型,随氯化钙浓度升高寒害表型逐渐加重;外施7 mmol/L氯化钙能够显著改善胡椒的净光合速率,随氯化钙浓度升高,净光合速率越来越低,氯化钙浓度过高时（达到28 mmol/L）净光合速率低于CK;与CK相比,外施7 mmol/L氯化钙能够显著提高抗氧化酶活性、增加可溶性糖含量、降低丙二醛含量。随处理浓度升高,抗氧化酶活性和可溶性糖含量越来越低,丙二醛含量越来越高。本研究结果为胡椒生产上抗寒技术指导和抗寒分子育种提供了参考。     

基于RNMU的多源星载SAR影像融合与土地覆盖分类

为充分利用多时相、多极化SAR数据在不同土地覆盖类型中的后向散射特性,将递归非负矩阵下近似(Recursive nonnegative matrix underapproximation,RNMU)算法引入多源SAR数据的融合,并利用融合后的SAR影像实现较高精度的土地覆盖分类。融合过程中,在根据不同模式SAR影像特点进行多源SAR影像预处理的基础上,基于RNMU算法通过对多个输入SAR影像进行矩阵分解及迭代最优矩阵求解,得到融合影像。为验证融合后SAR影像在土地覆盖分类中的应用效果,以吉林省大安市为研究区,对多时相Sentinel-1的VV/VH双极化SAR数据和高分三号(GF-3)的HH/HV双极化SAR数据进行了基于RNMU的影像融合,并利用融合后的SAR影像进行研究区主要土地覆盖类型分类。实验结果表明,基于RNMU融合影像的土地覆盖分类总体精度达93. 11%,Kappa系数为0. 86,与Gram-Schmid(G-S)融合方法相比,分类总体精度提高了6. 83个百分点,Kappa系数提高0. 12。多源SAR融合为SAR影像融合提供了有效手段,为土地覆盖分类提供了更多高精度的数据资源。     

Soluble Klotho protein suppresses THP-1-derived foam cell formation

AIMTo investigate the role of soluble Klotho protein in THP-1-derived foam cell formation. METHODSTHP-1 monocytes were induced into macrophages by treatment with 160 nmol/L phorbol myristate acetate for 48 h, and then were divided into 6 groups: negative control group (THP-1-derived macrophages), positive control group [THP-1-derived foam cells induced by oxidized low-density lipoprotein (ox-LDL) for 48 h], and 25, 50, 100 and 200 μg/L soluble Klotho protein groups (THP-1-derived macrophages pretreated with soluble Klotho protein at the indicat?ed concentraions for 2 h and then induced by ox-LDL for 48 h). Lipid droplets in cytoplasm were observed by oil red O staining. The cholesterol outflow rate was detected by scintillation counting technique. The content of intracellular total cholesterol, free cholesterol and cholesterol ester was detected by enzyme fluorescence analysis. The expression of acyl-coenzyme A:cholesterol acyltransferase 1 （ACAT1） and ATP-binding cassette transport?er A1 (ABCA1) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. RESULTSOil red O staining and lipid mass quantification showed that THP-1-derived foam cell formation was dose-dependently suppressed by soluble Klotho protein. The cholesterol efflux rate of THP-1-derived foam cells was increased by soluble Klotho protein in a dose-dependent manner (P<0.05). In addition, soluble Klotho protein decreased the expression of ACAT1 and increased the expression of ABCA1 in a dose-dependent manner (P<0.05). CONCLUSION The soluble Klotho protein inhibits THP-1-derived foam cell formation in a dose-dependent manner by down-regulating the expression of ACAT1 and up-regulating the expression of ABCA1.     

朱顶红无菌苗叶片高效再生体系

以朱顶红（Hippeastrum vittatum）叶片为外植体进行离体培养，具有取材方便、试材充足、成本低等优势，但叶片诱导再生率极低，是朱顶红离体培养的一大难题。本试验中分别以‘花孔雀’和‘黑天鹅’朱顶红无菌苗叶片为外植体，探究了不同植物生长调节剂和不同取材部位对不定芽诱导和继代增殖的影响。结果表明：最佳外植体为 MS 培养基中培养 10 d 形成的幼嫩叶片基部（0.5 cm），在光照 16 h · d-1（光照强度 36 μmol · m-2 · s-1）下，不定芽诱导的最适培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 NAA + 2 mg · L-1 TDZ，两个品种的不定芽均以间接途径发生，其中‘花孔雀’在培养 40 d 后形成愈伤组织，55 d形成不定芽，诱导率可达 69.44%；‘黑天鹅’在培养 45 d 后形成愈伤组织，65 d 形成不定芽，诱导率达到 66.67%；最适体细胞胚诱导培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 PIC，‘花孔雀’和‘黑天鹅’的诱导率分别达到 66.67%和 63.89%；最佳不定芽增殖培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 NAA + 1 mg · L-1 TDZ，‘花孔雀’和‘黑天鹅’的增殖系数分别达到 4.67 和 3.46；在不添加植物生长调节剂的 MS培养基中进行生根培养，30 d 后两个品种的生根率均达到 100%；将生根培养 30 d 的小植株转移至室温条件下放置 3 d，摘去封口膜再驯化 3 d 后，移栽至经高温消毒的草炭︰蛭石（体积比）为 1︰1 的基质中，成活率达到 100%