opaque 2基因微卫星标记与玉米籽粒赖氨酸含量的关系

利用opaque 2 (o2 )基因序列内的微卫星标记phi0 5 7、phi112和umc10 6 6检测优质蛋白和普通玉米自交系CA335及黄早四 ,发现phi0 5 7是共显性标记 ,且具有明显的多态性。利用标记phi0 5 7对BC1F1回交群体 [(CA335×黄早四 )×CA335 ]的 2 2 4个单株进行检测 ,两种基因型o2o2和o2O2的分离比为 1∶1 2 4 ,符合孟德尔遗传分离比例。进一步将群体内同一基因型o2O2或o2o2植株上的籽粒混合测定赖氨酸含量 ,发现o2o2基因型玉米籽粒的赖氨酸含量 (0 4 2 8% )显著大于o2O2基因型 (0 334% )。研究表明利用微卫星标记phi0 5 7辅助选择玉米o2o2基因型 ,可以提高优质蛋白玉米的选择效率。

糯玉米opaque2基因近等基因系的创制

opaque2基因能够提高糯玉米赖氨酸、色氨酸等必需氨基酸的含量。用2种优质蛋白玉米(QPM)CA339和鲁2548自交系作为opaque2基因供体,25个品质较好的糯玉米自交系作为受体,利用回交技术和SSR分子标记辅助选择育种,创制糯玉米opaque2近等基因系。分子标记结果表明,不同受体以及两种供体间都存在多态性。5套创制成功的opaque2近等基因系的赖氨酸含量比其轮回亲本分别提高了59.0%、52.7%、48.5%、46.3%和61.9%,分别由0.308%、0.313%、0.309%、0.341%、0.323%提高到0.489%、0.478%、0.458%、0.498%、0.522%。本研究表明,可以利用此方法通过向不同遗传背景的多种受体导入opaque2,选取赖氨酸含量提高较大、透明表型的近等基因系,提高糯玉米的营养价值和经济价值。

Opaque2转录因子对玉米α-醇溶蛋白基因家族成员表达的影响

醇溶蛋白是玉米主要的储存蛋白,其中α-醇溶蛋白含量最为丰富,对玉米籽粒品质有重要影响。本研究利用全基因组数据鉴定α-醇溶蛋白基因家族,并从基因保守motif、玉米籽粒不同发育时期基因的表达谱以及转录因子Opaque2对该家族成员表达的影响3个方面分析玉米α-醇溶蛋白基因家族。结果显示,玉米参考基因组中存在39个α-醇溶蛋白基因,其中z1A、z1B、z1C和z1D 4个亚族成员分别为12个、8个、14个和5个。基因保守motif分析发现该家族基因序列高度保守。表达谱分析结果显示该家族表达模式差异较大,其中z1A亚族在籽粒发育时期表达量最高。利用RNA-seq数据剖析在种子发育阶段Opaque 2(O2)野生型和突变体编码该家族成员的表达差异,结果显示O2突变体中z1C亚族成员表达显著下调,z1A亚族表达略有下调,z1B和z1D亚族成员表达量没有显著的变化。本研究在重新注释α-醇溶蛋白家族成员信息的基础上,对该家族的表达谱进行了分析,为今后玉米籽粒品质育种提供了必要遗传信息。

RNAi靶向沉默opaque2基因对高粱醇溶蛋白含量的影响

为研究opaque2基因沉默对高粱醇溶蛋白含量的影响,采用RNAi技术构建opaque2基因抑制表达载体,利用农杆菌介导转化高粱幼胚,测定转基因植株籽粒中醇溶蛋白含量,SDS-PAGE电泳检测醇溶蛋白带型,并使用透射电镜观察胚乳细胞中蛋白质体变化。结果表明,与野生型相比,5株转基因植株中4株籽粒的醇溶蛋白含量显著降低(P<0.05);野生型和各转基因植株的醇溶蛋白SDS-PAGE电泳带型相同;野生型胚乳细胞中淀粉粒排列紧密,部分大淀粉粒融合成大块状,蛋白质体数量较多且有一些体积较大;而转基因植株淀粉粒排列疏松,细胞中存在多处孔隙,蛋白质体数量较少,体积较小。opaque2基因沉默引起胚乳细胞蛋白质体数量变少,体积变小,进而影响胚乳细胞内部结构和籽粒醇溶蛋白含量。

A white opaque substance-positive gastric hyperplastic polyp with dysplasia

The endoscopic findings of gastric hyperplastic polyps (HPs) with dysplasia have not been well-defined, and the clinical significance of these lesions, including their malignant potential, is unclear. In this report, we describe a case of a white opaque substance (WOS)positive gastric HP with dysplasia. A 76-year-old woman was referred to our hospital for endoscopic resection of a gastric HP. Upper endoscopy revealed a 25-mm whitish and reddish polypoid lesion on the greater curvature in the lower third of the stomach. The whitish part was diagnosed as a WOS using conventional and magnifying endoscopy with narrow band imaging. An examination of the biopsy specimen indicated that the lesion was a typical gastric HP. However, because of its color and the presence of a WOS, we suspected that this lesion was an atypical gastric HP. Therefore, we performed a polypectomy. Histopathologically, diffuse lowto high-grade dysplasia was found on the surface of the polyp. We performed immunohistochemical staining using a monoclonal antibody specific for adipophilin as a marker of lipid droplets (LDs). LDs were detected in approximately all of the neoplastic cells, especially in the surface epithelium of the intervening apical parts and were located in the subnuclear cytoplasm of the neoplastic cells. According to endoscopic and histopathological findings, the WOS-positive epithelium indicated dysplasia of the gastrointestinal phenotype, which could absorb lipids. The presence of a WOS in a gastric HP may be considered an endoscopic finding that is predictive of the neoplastic transformation of a gastric HP. We suggest that a WOS-positive gastric HP should be resected endoscopically to investigate its neoplastic transformation.

通过荧光标记的凝胶阻滞技术分析Opaque2蛋白与ZmGRAS11启动子的结合位点

凝胶阻滞实验(electrophoretic mobility shift assay,EMSA)是研究蛋白质与核酸结合的一种关键实验技术。EMSA技术兴起以来,使用放射性同位素、生物素标记核酸探针的手段已经非常成熟,但这两种传统的标记技术分别具有放射性探针稳定性差和生物素检测步骤复杂等缺点。近年来,尽管荧光标记探针逐渐被应用于EMSA中,但是对于利用荧光标记探针的EMSA仍缺乏系统的报道。对荧光标记的EMSA技术流程进行了优化和系统总结;利用6-羧基荧光素(6-carboxy-fluoroscine,FAM)标记ZmGRAS11启动子探针,通过EMSA检测其与Opaque2蛋白的结合,明确了蛋白和探针的适宜比例为8∶1。对GCN4 motif序列碱基进行突变并利用EMSA分析Opaque2与ZmGRAS11启动子之间的结合位点,结果表明GCN4 motif的“TGAC”核心基序在ZmGRAS11启动子与Opaque2蛋白的结合中可能起到了关键作用。研究结果为进一步探究Opaque2-ZmGRAS11转录调控模块在玉米籽粒发育中的作用机理提供了数据支撑。

Paired eye-control study of unilateral opaque bubble layer in femtosecond laser assisted laser in situ keratomileusis

AIM: To investigate the predictive factors of the opaque bubble layer(OBL) compared to the fellow eye of same patients in FS200 femtosecond laser assisted laser in situ keratomileusis(FS-LASIK).METHODS: This study consisted of 60 consecutive patients(120 eyes) with unilateral OBL during FS-LASIK. Eyes were divided into OBL(the OBL eyes) and OBL-free groups(the fellow eyes) based on the occurrence of OBL. The preoperative demographic data, refraction, keratometry, corneal astigmatism, pachymetry, intraocular pressure and intraoperative data including the outlet location of gas diffusing canal were collected. Conditional logistic regression analysis was performed to find the associated factors with OBL in the two groups by determining odds ratios(OR) and 95%CI.RESULTS: The preoperative demographic data, mean spherical errors, mean K value, suction time, intraocular pressure and central cornea thickness were not significantly different between the two groups. The outlet location of gas diffusing canal(P<0.01, OR 7.16, 95%CI 2.53-20.32) and the corneal astigmatism(P=0.013, OR 0.13, 95%CI 0.03-0.65) were significantly associated with the incidence of OBL by multivariate logistic regression analysis. Visual acuity, efficacy, and safety were comparable between the two groups two months after surgery except for a slightly lower predictability value for the hard OBL eyes.CONCLUSION: The reduction of the incidence of OBL is obvious when the outlet of gas diffusing canal located at the posterior border of the corneoscleral limbus. This is probably consequent to more effectiveness of gas diffusing canal. Corneal astigmatism is also an independent protective factor for OBL formation.

Beneficial effects of vincamine with thioctic acid and lutein on retinal and optic nerve functions in an opaque media

AIM: To detect whether the combination of vincamine, thioctic acid and lutein will improve the retina and optic nerve functions in cases of an opaque media with an optic nerve and/or a retinal defect or not.METHODS: Totally 2000 patients(2000 eyes) of corneal opacities with defects in the optic nerve or/and the retinal functions were studied. Every patient received three types of drugs: thioctic acid with cyanocobalamine, vincamine, and lutein. The drugs were given daily for 3-12 mo according to patient’s responses. Full field flash electroretinogram(ERG) and flash visual evoked potential(VEP) were done before treatment and at 1, 3, 6, and 12 mo sequentially. Patients were followed up for 12 mo.RESULTS: In the 2000 eyes, 1000 eyes had both moderate optic nerve and retinal function defects;and 840 eyes out of the 1000 improved with the medical treatment. Another 500 eyes out of the 2000 eyes had extinguished retinal function with normal optic nerve function and only 125 eyes of them improved. The 290 out of the 2000 eyes had severe defects in optic nerve with normal retinal function and 130 of them improved. Another 210 eyes have mild optic nerve and retinal function defects and 194 improved.CONCLUSION: The combination of vincamine, thioctic acid with cyanocobalamine, and lutein improved both retina and optic nerve functions mainly in mild and moderate defect than in severe cases.

Association between white opaque substance under magnifying colonoscopy and lipid droplets in colorectal epithelial neoplasms

AIM To examine the association between white opaque substance(WOS) and histologically verified lipiddroplets in colorectal epithelial neoplasms.METHODS We reviewed colonoscopy records at our institution from 2014 to 2016 and identified cases of endoscopically or surgically resected colorectal epithelial neoplasms observed by magnifying narrow-band imaging(M-NBI) colonoscopy. Immunohistochemistry was used to stain tumors with a monoclonal antibody specific to adipophilin as a marker of lipids. The expression and distribution of adipophilin were compared between WOS-positive and WOS-negative lesions and among tumors classified by histologic type and depth of invasion.RESULTS Under M-NBI colonoscopy, 81 lesions were positive for WOS and 48 lesions were negative for WOS. The rate of adipophilin expression was significantly higher in WOS-positive lesions(95.1%) than in WOS-negative lesions(68.7%)(P = 0.0001). The incidence of deep adipophilin expression was higher in WOS-positive lesions(24.7%) than in WOS-negative lesions(4.2%)(P = 0.001). The incidence of deep expression was predominant among cancers with massive submucosal invasion(62.5%) compared to adenoma(7.2%) and high-grade dysplasia or cancers with slight submucosal invasion(12.7%)(P = 0.0001).CONCLUSION The distribution of lipid droplets may be closely associated with the visibility of WOS under M-NBI colonoscopy, and with histologic grade and depth of tumor invasion.

Sucrose-associated SnRK1a1-mediated phosphorylation of Opaque2 modulates endosperm filling in maize

During maize endosperm filling,sucrose not only serves as a source of carbon skeletons for storage-reserve synthesis but also acts as a stimulus to promote this process.However,the molecular mechanisms underlying sucrose and endosperm filling are poorly understood.In this study,we found that sucrose promotes the expression of endosperm-filling hub gene Opaque2(O2),coordinating with storage-reserve accumulation.We showed that the protein kinase SnRK1a1 can attenuate O2-mediated transactivation,but sucrose can release this suppression.Biochemical assays revealed that SnRK1a1 phosphorylates O2 at serine 41(S41),negatively affecting its protein stability and transactivation ability.We observed that mutation of SnRK1a1 results in larger seeds with increased kernel weight and storage reserves,while overexpression of SnRK1a1 causes the opposite effect.Overexpression of the native O2(O2-OE),phospho-dead(O2-SA),and phospho-mimetic(O2-SD)variants all increased 100-kernel weight.Although O2-SA seeds exhibit smaller kernel size,they have higher accumulation of starch and proteins,resulting in larger vitreous endosperm and increased test weight.O2-SD seeds display larger kernel size but unchanged levels of storage reserves and test weight.O2-OE seeds show elevated kernel dimensions and nutrient storage,like a mixture of O2-SA and O2-SD seeds.Collectively,our study discovers a novel regulatory mechanism of maize endosperm filling.Identification of S41 as a SnRK1-mediated phosphorylation site in O2 offers a potential engineering target for enhancing storage-reserve accumulation and yield in maize.

玉米品质相关的opaque基因研究进展

玉米中的opaque突变体改变了胚乳的蛋白特性,导致胚乳表现柔软且不透明的粉质状。粉质胚乳的高赖氨酸营养特性引起人们的极大关注,研究人员先后发现了13个opaque胚乳突变体,只有o2的分子机理研究较为清楚,对提高赖氨酸含量的作用最大。为了研究胚乳中储藏物质合成、装配、转运的调控机理,从而将这些有益突变基因应用于农业生产,研究人员继而发现了多个胚乳修饰的主效位点及基因(Opm)。本文综述玉米opaque突变体及相关基因的研究进展,对当前育种中利用该类基因培育优质蛋白玉米(QPM)的研究状况进行分析,为高赖氨酸玉米的分子标记辅助选择(MAS)和基因聚合育种提供参考。

郑58/opaque2近等基因系中opaque2基因突变位点分析与高效分子标记开发

opaque2突变体材料是最常用的高赖氨酸玉米供体。对已获得的郑58/o2近等基因系研究表明,胚乳发育不同时期22-kDα-醇溶蛋白的积累均明显低于郑58。荧光定量PCR结果表明,α-醇溶蛋白家族基因Z1A、Z1B、Z1C和Z1D的表达均显著低于郑58。郑58/o2胚乳发育不同时期Opaque2基因均正常表达。测序分析发现,郑58/o2中o2基因ATG后713 bp处缺失10个碱基,ORF预测Opaque2蛋白翻译提前终止。针对突变缺失位点开发基因内分子标记o2-indel-1,利用该标记进行回交转育,结果表明,o2-indel-1与o2突变表型完全连锁,错选率为0。opaque2基因突变位点的解析有助于高效分子标记的开发,有效降低错选率,提高优质蛋白鲜食玉米多基因聚合育种的选择效率。

玉米opaque2近等基因系中相关基因的表达量分析

Opaque2(O2)基因编码产生OPAQUE2蛋白,该蛋白为碱性亮氨酸拉链家族的一种转录因子,可作用于基因的启动区,调控基因的转录。辽2345/o2为本实验室构建的辽2345的opaque2(o2)基因回交导入系,蛋白组学分析表明,辽2345与辽2345/o2在授粉后18 d的胚乳细胞中表达蛋白的种类和数量存在很大差异,从中筛选出差异较明显的9种蛋白质。为了进一步验证O2与9种蛋白间的作用关系,应用相对荧光定量PCR的方法对9种蛋白进行了RNA水平表达量的检测。结果表明,在胚乳发育过程中Chfi很可能受到O2的抑制作用,O2很可能对Ubc、Lgl、Sdh、LOC541920、Shmt、Ppdk等基因存在某种促进或激活作用。

opaque2对糯玉米子粒食味和营养品质的影响分析

玉米opaque2(o2)是胚乳发育的核心调控基因,对子粒发育至关重要。基于9组糯玉米o2近等基因系(o2-NILs)及对应轮回亲本,对鲜食期和成熟期子粒的种皮厚度、总淀粉含量、可溶性糖含量等性状进行分析。结果表明,黄糯2/o2o2和中63-2/o2o2鲜食期子粒的食味品质性状均有提高,其他o2-NILs子粒品质变化不一。鲜食期o2突变对种皮厚度影响较大,但与成熟期无直接相关性。o2突变使玉米子粒赖氨酸含量增加,营养价值提高,但多数近等基因系成熟期淀粉含量和百粒重降低,仅糯2/o2o2、淀粉含量和百粒重均不受影响。糯2/o2o2、SY1-2/o2o2、赵OP-6/o2o2中27 KDγ-zein表达量显著增加、子粒饱满度与轮回亲本相近,但均表现为粉质胚乳,因此认为,优质蛋白糯玉米硬质胚乳的恢复需多个o2胚乳修饰基因协同作用。

OPAQUE3, encoding a transmembrane bZIP transcription factor, regulates endosperm storage protein and starch biosynthesis in rice

Starch and storage proteins are the main components of rice(Oryza sativa L.)grains.Despite their importance,the molecular regulatory mechanisms of storage protein and starch biosynthesis remain largely elusive.Here,we identified a rice opaque endosperm mutant,opaque3(o3),that overaccumulates 57-kDa proglutelins and has significantly lower protein and starch contents than the wild type.The o3 mutant also has abnormal protein body structures and compound starch grains in its endosperm cells.OPAQUE3(O3)encodes a transmembrane basic leucine zipper(bZIP)transcription factor(OsbZIP60)and is localized in the endoplasmic reticulum(ER)and the nucleus,but it is localized mostly in the nucleus under ER stress.We demonstrated that O3 could activate the expression of several starch synthesis-related genes(GBSSI,AGPL2,SBEI,and ISA2)and storage protein synthesis-related genes(OsGluA2,Prol14,and Glb1).O3 also plays an important role in protein processing and export in the ER by directly binding to the promoters and activating the expression of OsBIP1 and PDIL1-1,two major chaperones that assist with folding of immature secretory proteins in the ER of rice endosperm cells.High-temperature conditions aggravate ER stress and result in more abnormal grain development in o3 mutants.We also revealed that OsbZIP50 can assist O3 in response to ER stress,especially under high-temperature conditions.We thus demonstrate that O3 plays a central role in rice grain development by participating simultaneously in the regulation of storage protein and starch biosynthesis and the maintenance of ER homeostasis in endosperm cells.

ZmGRAS11,transactivated by Opaque2,positively regulates kernel size in maize

Although the genetic basis for endosperm development in maize(Zea mays)has been well studied,the mechanism for coordinating grain filling with increasing kernel size remains elusive.Here,we report that increased kernel size was selected during modern breeding and identify a novel DELLA-like transcriptional regulator,ZmGRAS11,which positively regulates kernel size and kernel weight in maize.We find that Opaque2,a core transcription factor for zein protein and starch accumulation,transactivates the expression of Zm GRAS11.Our data suggest that the Opaque2-Zm GRAS11 module mediates synergistic endosperm enlargement with grain filling.

Petrogenesis of opaque assemblages in the Ningqiang carbonaceous chondrite

Numerous round to oblate opaque assemblages (OAs) are found in chondrules and matrix of the Ningqiang carbonaceous chondrite. They are mainly composed of Ni-rich metal,magnetite,Fe,Ni-sulfides,with minor amounts of phosphate,phosphoran-olivine,pyroxene and trace amounts of nano-sized platinum-group metal alloys. The mineralogy of Ningqiang OAs is very similar to that of OAs previously reported in Ca,Al-rich inclusions of CV chondrites. Being a rare mineral phase in nature,phosphoran-olivine is thought to form by nonequilibrium reactions between P-bearing molten metal and olivine crystals during rapid cooling. Its occurrence in Ningqiang OAs indicates that the precursor of OAs was locally produced during chondrule formation,rather than directly condensed from the solar nebula as previously thought. The petrographic and mineralogical characteristics of Ningqiang OAs reveal that OAs formed by low temperature alterations of pre-existing homogeneous alloys within chondrules on a planetary body.

Higher concentration of CO2 and 37℃ stabilize the less virulent opaque cell of Candida albicans

Background Candida albicans （C. albicans） strains can spontaneously switch at a very low frequency from white to opaque phase. The ability to switch reversibly between white and opaque phenotype and contributes to the pathogenicity of C. albicans. White and opaque switching can be induced by various environmental signals. Previous study showed that opaque cells switch en masse to white when transferred in vitro to 37℃, the temperature of their animal host. The objective of the present study was to determine the effect of different concentration of carbon dioxide and temperature on white-opaque switching, and to determine the different anti-candida killing activity of white and opaque form by human monocyte-macrophage cell line THP-1.Methods White-opaque switching and opaque-white switching were assayed. Modified Lee＇s medium supplemented with phloxine B was used to detect white and opaque forms of C. albicans under 0.03% CO2 at 25℃, 0.03% CO2 at 37℃ and 5% CO2 at 37℃. Growth curve of C. albicans was monitored using OD value at 630 nm simultaneously. White and opaque forms of C. albicans and THP-1 cells were cocultured at ratio of 1：10. Colony serial dilutions were used to assay for intracellular candidacidal activity. MTT assay was used to measure the extracellular candidacidal activity.Results Phenotype switching was successfully induced in vitro in all three strains of C. albicans. When evaluating white to opaque switching, opaque colony proportion of all colonies was 0.572±0.087, 0.920±0.030 and 0.985±0.026 exposure of white cells to 0.03% CO2 at 25℃, 0.03% CO2 at 37℃ and 5% CO2 at 37℃. When evaluating opaque to white switching, opaque colony proportion of all colonies was 0.600±0.114, 0.983±0.003 and 0.998±0.003 exposure of white cells to 0.03% CO2 at 25℃, 0.03% CO2 at 37℃ and 5% CO2 at 37℃. No significant difference of white or opaque form growth rate was found among three conditions （P〉0.05）. THP-1 mediated extracellular anti-candida activity in white form was （79.80±3.71）% and （56.28±19.12）% at different dilution ratio, which were significantly lower than that in opaque form （100%, P 〈0.01）. THP-1 mediated intracellular anti-candida activity in white form （（62.98±5.02）%） was significantly lower than that in opaque form （（87.07±1.80）%, P 〈0.01）.Conclusions Our results showed that opaque form is more vulnerable and less virulent than that in white form. It suggested that higher concentration of CO2 and 37℃ in host niches stabilize the less virulent opaque cell of C. albicans,which might have implications for pathogenesis, commensalism and mating.

Fabrication of opaque and transparent 3D structures using a single material via twophoton polymerisation lithography

Two-photon polymerisation lithography enables the three-dimensional(3D)-printing of high-resolution micron-and nano-scale structures.Structures that are 3D-printed using proprietary resins are transparent and are suitable as optical components.However,achieving a mix of opaque and transparent structures in a single optical component is challenging and requires multiple material systems or the manual introduction of ink after fabrication.In this study,we investigated an overexposure printing process for laser decomposition,which typically produces uncontrollable and random‘burnt’structures.Specifically,we present a printing strategy to control this decomposition process,realising the on-demand printing of opaque or transparent structures in a single lithographic step using a single resin.Using this method,opaque structures can be printed with a minimum feature size of approximately 10μm,which exhibit<15%transmittance at a thickness of approximately 30μm.We applied this process to print an opaque aperture integrated with a transparent lens to demonstrate an improved imaging contrast.