鲜食番茄茄红素基因的AFLP-SCAR分子标记

番茄红素(Lycopene)是功能性天然色素,因其高抗氧化功能而备受关注.为筛选高番茄红素品种,加速鲜食番茄分子标记辅助育种进程,本试验以高茄红素番茄骨干系F-516F8和低茄红素骨干系Nor作为试验材料,研究了鲜食番茄高茄红素基因的AFLP-SCAR分子标记.利用杂交F1代、自交F2代遗传群体,通过64对E/M引物组...     

茄红素可藉由调降NF-kB及Sp1并降低基质金属蛋白酶-9表现而抑制人类肝癌细胞株SK-Hep-1侵袭(英文)

目的:近年来许多研究显示,摄取较多富含茄红素的食物可降低摄护腺癌以及肝癌的发生率。虽然茄红素被证实具有抑制癌症转移的能力,但目前为止其作用机制仍不清楚。因此,本研究的主要目的在于探讨茄红素的抗癌转移作用及机制。我们提出的假说为:茄红素具有抑制癌转移的能力,其机制可能与调降基质金属蛋白酶-9(matrixmetalloproteinase-9;MMP-9)的表现有关。试验方法:将高侵袭能力的肝癌细胞株SK-Hep-1以茄红素预培养2h,以磷酸盐类缓冲溶液清洗后并继续培养24h,之后进行各种分析,包括:以transwell分析癌细胞侵袭能力;以同功酵素图谱(zymography)及西方墨点法个别分析MMP-9的酵素活性及蛋白表现;另外,利用电泳移动偏移分析(electrophoreticmobilityshiftassay;EMSA)检测转录因子NF-kB、AP-1以及Sp1对MMP-9启动子(promoter)的结合能力。结果:茄红素具有抑制肝癌细胞侵袭的能力,而抑制效果与剂量关系呈钟型曲线,亦即以5mmol/L剂量的抑制效果最好(抑制率达63%,P<0.001);同时,在抑制MMP-9的活性及蛋白表现上也呈相同趋势。此外,茄红素也会降低转录因子NF-kB及Sp1对MM-9启动子的结合能力。结论:茄红素具有抑制癌细胞侵袭的能力,其机制可能与降低转录因子NF-kB和Sp1结合至MMP-9启动子的能力以及减少MMP-9蛋白的表现有关。本研究增加了对茄红素抑制癌转移作用机制的了解,将有助于茄红素作为保健食品上的实用基础。     

番茄茄红素β-环化酶基因高效沉默载体构建

番茄茄红素是番茄果实中含量最多的一种类胡萝卜素,也是植物类胡萝卜素代谢过程中的一个重要中间产物.它的环化是在茄红素β-环化酶(Lyc-b)和茄红素ε-环化酶(Lyc-e)催化下完成的,其中茄红素β-环化酶在番茄茄红素转变为β-胡萝卜素的过程中起主要作用.我们利用RT-PCR方法从番茄叶片中克隆出Lyc-b基因,将PCR产物与pGEM-T载体连接后进行测序,扩增的DNA序列与发表的茄红素β-环化酶序列一致.以此为模板,克隆了400 bp左右的目的片段,利用Gateway系统构建Lyc-b基因目的片段的pJawoh18-2b ihpRNA表达载体.经PCR与酶切鉴定的阳性克隆转化农杆菌LBA1100,利用农杆菌叶盘法转化番茄获得了抗性愈伤及再生苗,以检测ihpRNA沉默载体能否降低茄红素β-环化酶基因表达,并为进一步了解该基因在番茄类胡萝卜素代谢过程中的作用奠定基础.这些数据未在本文列出.     

茄红素可藉由调降NF—κB及Sp1并降低基质金属蛋白酶-9表现而抑制人类肝癌细胞株SK—Hep-1侵袭

目的：近年来许多研究显示，摄取较多富含茄红素的食物可降低摄护腺癌以及肝癌的发生率。虽然茄红素被证实具有抑制癌症转移的能力，但目前为止其作用机制仍不清楚。因此，本研究的主要目的在于探讨茄红素的抗癌转移作用及机制。我们提出的假说为：茄红素具有抑制癌转移的能力，其机制可能与调降基质金属蛋白酶-9（matrix metalloproteinase-9；MMP-9）的表现有关。试验方法：将高侵袭能力的肝癌细胞株SK—Hep-1以茄红素预培养2h，以磷酸盐类缓冲溶液清洗后并继续培养24h，之后进行各种分析，包括：以transwell分析癌细胞侵袭能力；以同功酵素图谱（zymography）及西方墨点法个别分析MMP-9的酵素活性及蛋白表现；另外，利用电泳移动偏移分析（electrophoretic mobility shift assay；EMSA）检测转录因子NF—κB、AP-1以及Sp1对MMP-9启动子（promoter）的结合能力。结果：茄红素具有抑制肝癌细胞侵袭的能力，而抑制效果与剂量关系呈钟型曲线，亦即以5mmol／L剂量的抑制效果最好（抑制率达63％，P〈0．001）；同时，在抑制MMP-9的活性及蛋白表现上也呈相同趋势。此外，茄红素也会降低转录因子NF—κB及Sp1对MM-9启动子的结合能力。结论：茄红素具有抑制癌细胞侵袭的能力，其机制可能与降低转录因子NF—κB和Sp1结合至MMP-9启动子的能力以及减少MMP-9蛋白的表现有关。本研究增加了对茄红素抑制癌转移作用机制的了解，将有助于茄红素作为保健食品上的实用基础。     

梅花鹿鹿茸真皮层细胞的分离培养及冷冻保存

为了研究梅花鹿鹿茸真皮层细胞的生物学特性,取梅花鹿鹿茸生长顶端的真皮层组织,分离真皮层细胞进行体外培养及冷冻保存。结果表明,在含10 %胎牛血清(FBS)的DMEM培养基中,鹿茸真皮层细胞能进行短期体外培养,培养的细胞呈长梭形或菱形,细胞的生长状况良好,传代培养的细胞培养5d可长至汇合。传4代以前的细胞形态均一,边缘光滑,胞质均匀透明,细胞排列紧密;传5代后,细胞伸展变为扁平形,细胞突起增多,边缘不光滑,汇合后细胞排列疏松。以含5%二甲基亚砜(DMSO)和10%FBS的DMEM为冻存液,经梯度降温后冻存,真皮层细胞复苏后的存活率较高。     

牛滋养层干细胞样细胞系的建立

为了成功建立牛滋养层干细胞样细胞系,探讨牛滋养层干细胞样细胞（Bovine Trophoblastic Stem-like Cells）体外培养条件及相关影响因素,以胎牛子宫成纤维细胞条件培养液作为培养基,胶原铺被培养皿,利用体外受精牛囊胚培养得到牛滋养层干细胞样细胞,并调整培养体系,以探讨其最适生长条件。结果表明：利用添加FBS、β-巯基乙醇、肝素以及胎牛子宫成纤维细胞条件培养液的DMEM/F12,体外培养牛囊胚,可以初步建立牛滋养层干细胞样细胞系。     

不同发育阶段‘文紫椒1号’果色色素的比例和组织定位

研究旨在阐明‘文紫椒1号’果实在不同发育阶段的色素比例和组织定位,为丘北辣椒在观赏园艺中的应用和观赏型新奇果色新品种的选育提供参考。以种植于云南文山州砚山县的‘文紫椒1号’果实为试材,将果实分为5个发育阶段,用分光光度法检测了果实的叶绿素含量([Chl])、类胡萝卜素含量([Car])、总黄酮类含量(TFC)、总花色苷含量(TAC)和辣椒红素含量([Cap]),并分析其配比和相关性,同时用徒手切片法检测了色素的组织定位。结果表明,在发育过程中,‘文紫椒1号’果实[Chla]/[Chlb]的最大值出现在发育阶段3,[Chl(a+b)]/[Car]、[Chl(a+b)]/TFC和[Chl(a+b)]/TAC的最大值均出现在阶段2,而[Chl(a+b)]/[Cap]、TFC/TAC、TFC/[Cap]和TAC/[Cap]的最大值均出现在阶段1。[Chla]、[Chlb]、[Chl(a+b)]、[Car]、TFC、TAC和[Cap]之间的相关性不尽一致,其中,TAC与[Chl(a+b)]、TFC之间均呈不显著正相关,而与[Car]之间呈不显著负相关,[Cap]则与[Chl(a+b)]、[Car]、TFC和TAC之间分别呈显著负相关、极显著正相关、不显著正相关和不显著负相关。果实色素主要定位在中果皮细胞中,细胞的色相和色度变化趋势与果色的高度一致。所以,‘文紫椒1号’的果色源于各发育阶段果皮细胞中叶绿素、类胡萝卜素（含辣椒红素）和黄酮类（含花色苷）色素的比例差异。     

芒果MYB转录因子的鉴定及分析

MYB是植物重要的转录因子,对果实的发育发挥重要的作用。本研究基于芒果果实和叶片转录组数据,运用Nr、Swiss-prot和Pfam数据库进行Unigene的功能注释,共鉴定得到125个MYB家族转录因子,其中包含4R-MYB (1)、R1R2R3-MYB (1)、R2R3-MYB (51)、R1-MYB (8)和atypical-MYB (64)。通过在线数据库分析表明具有R重复区的共61个MYB蛋白中大部分属于亲水性蛋白,且均不含有信号肽和跨膜结构,亚细胞定位于细胞核。序列比对和进化树分析表明:芒果MYB转录因子的结构域具有高度保守性,结构分析含有[W]-X (19)-[W]-X (19)-[W]结构。最后通过GO功能注释得到大部分的MYB具有结合和转录调节活性。除此之外,MYB还可以具有催化活性和转运活性。在生物过程中,主要以细胞过程、生物调节、生物调节过程、刺激响应和代谢过程居多。本研究为进一步挖掘和分析芒果MYB转录因子功能奠定了基础。     

不同来源牛体外胚胎与牛子宫内膜细胞共培养体系的建立

为了初步建立有效的牛胚胎与子宫内膜细胞的体外共培养体系。本实验分别将体外受精第7天后的胚胎和孤雌激活,与培养7天的胚胎与子宫内膜细胞共培养,在48 h和72 h后分别统计体外受精实验组和孤雌激活实验组的孵化率。实验结果表明：在本实验室培养条件下,在与子宫内膜细胞共培养48 h后,体外受精胚胎和孤雌发育胚胎在孵化率上存在显著差异(40±5)%、(25±5)%,(P＜0.05);与子宫内膜共培养72 h后,孵化率无显著差异(70±8.66)%,(65±5)%,(P＞0.05)。本实验证明了在体外环境下,牛体外胚胎与子宫内膜细胞共培养后能够正常孵化,可以初步建立起牛体外胚胎与子宫内膜细胞的体外共培养体系,并为进一步研究该体系对牛体外胚胎培养质量的影响奠定一些基础。     

奶牛乳腺上皮细胞体外培养条件优化的初步研究

从培养基、细胞接种浓度、冻存液三方面对奶牛乳腺上皮细胞体外培养条件进行了优化。采用台盼兰染色计数方法绘制细胞生长曲线,评定细胞体外生长增殖。结果表明,使用DMEM/F12培养基细胞生长状况最好,DMEM培养的细胞较DMEM/F12生长缓慢,F12其次,RPMI1640培养细胞生长最慢;以1×104的接种浓度于24孔板,细胞生长状态最好,细胞的生长周期经历了潜伏期、对数生长期、稳定期、衰亡期4个生长阶段,生长曲线符合"S"型生长规律;采用胎牛血清(90%FBS+10%DMSO)和培养基(70%培养基+20%FBS+10%DMSO)两种冻存液冻存奶牛乳腺上皮细胞,细胞复苏培养后,胎牛血清冻存的细胞生长状态和增殖速度都明显优于培养基冻存的奶牛乳腺上皮细胞,从培养的第3天开始,2组细胞数量差异显著(P0.05)。本研究结果为建立体外培养体系提供了一定参考。     

Quantifying lycopene synthesis and chlorophyll breakdown in tomato fruit using remittance VIS spectroscopy

The aim of this study was to increase the understanding of chlorophyll breakdown and lycopene synthesis at a quantitative level in Solanum lycopersicum fruit. To accomplish this, a kinetic model is proposed describing the transition from chloro- to chromoplast. Remittance VIS spectroscopy was used to assess chlorophyll and lycopene levels non-destructively in cocktail and round type tomatoes. Tomatoes were stored at constant temperatures between 4 and 24 °C, or at a stepwise changing temperature between 4 and 16 °C. Chlorophyll and lycopene levels were measured repeatedly over time and used to calibrate a kinetic model that describes how an autocatalytic enzyme system links chlorophyll breakdown to lycopene synthesis, including breakdown of lycopene precursor and lycopene itself. Increasing storage temperatures increased the reaction constant for lycopene synthesis more than that of chlorophyll breakdown for both tomato types. The reaction constants describing chlorophyll breakdown and lycopene synthesis were considerably larger, and the estimated enzyme levels lower for the round type. This allows round tomatoes to quickly resume lycopene synthesis after a cold storage period when enzyme levels are low. Lycopene breakdown was established for the round type while the cocktail type showed lycopene precursor breakdown. Chlorophyll breakdown and lycopene synthesis, as affected by storage temperature and tomato type, is covered well by the model for both tomato types. We hypothesise that the postulated enzyme system, responsible for the direct link between chlorophyll breakdown and lycopene synthesis, is due to STAY-GREEN proteins. Remittance VIS spectroscopy is, in combination with a parameter estimation tool, suited to screen tomato genotypes for intended colour transformation performance, or as tool in chloroplast to chromoplast transition studies.     

番茄红素的提取及生理功能的研究

番茄红素为开链式不饱和类胡萝卜素,具有抗氧化、清除自由基、促进细胞间连接与传导、防癌抗癌等多种生理功能。作为一种功能性天然色素,番茄红素被广泛应用于多种功能性食品。从分子结构、提取生产以及功能性质方面介绍了番茄红素的发展情况。     

番茄红素快速测定方法的研究

以番茄红素含量不同的58个基因型为材料,对CR400型色差计的快速测定值与番茄红素含量之间的相关性进行了研究。结果表明,以果实为样品测定的L1,a1,b1值在基因型间呈随机分布,番茄红素含量与以果浆为样品测定的L2和b2值呈负相关,与a2值呈极显著正相关。以去除自由水后的果酱为样品测定的L3,a3,b3值更加稳定, 它们与番茄红素含量的相关性更加密切。自由水含量对色差计的读数值影响较大,在自由水含量适中(71．11％- 76．20％)的基因型中,番茄红素含量与(a3／b3 a3／L3)的相关系数高达0．808,此时用线性回归方程能够比较准确地根据色差计的读数值估测果实中番茄红素的含量。     

鲜食番茄果肉中番茄红素含量性状的遗传分析

为更快更好地进行番茄果肉颜色的评定,定位相关QTLs以改良果肉的颜色品质,本研究选用果肉番茄红素含量显著不同的2个番茄品系,利用分光光度计法测定番茄红素含量,并通过P1、P2、F1、F2、B1和B2六世代联合分析法,研究分析了果肉番茄红素含量的遗传规律。结果表明：F2、B1、B2分离世代群体呈双峰或单峰偏态分布,主基因的加性效应明显,多基因对番茄红素含量分布有较大影响,存在明显的主基因多基因特征,显示番茄果肉番茄红素含量的遗传为多基因控制的数量性状;果肉番茄红素含量的遗传符合一个加性主基因和加性-显性多基因模型(D-2模型)。主基因遗传率较高,为27.39%~66.93%;多基因遗传率较低,为12.09%~48.44%。所以,利用有性杂交育种提高和固定高番茄红素性状,在早期世代进行选择有利于发现新QTLs位点。     

Spatial-temporal analyses of lycopene and sugar contents in tomatoes during ripening using chemical shift imaging

During the ripening process, fruit and vegetables undergo physico-chemical changes which affect their color, texture, aroma, and sugar content, etc. For tomatoes, color change is the main index of ripening stages, and it is associated with metabolized lycopene. In order to detect the internal changes of sugar and lycopene contents in tomatoes during ripening, chemical shift imaging (CSI) and high performance liquid chromatography (HPLC) analyses were employed. Imaging experiments on tomatoes were performed in a Bruker 7T MRI system using the gradient-echo sequence for scanning. Spatial distribution of the internal sugar and lycopene contents of tomatoes were, for the first time, revealed and compared before and after the ripening process. The CSI analysis showed that a linear relationship existed between CSI intensity and concentrations of sugar solutions in the range of 0.1-1.0 M. The CSI results indicate that there was no consistent trend in the variation of sugar signal intensity during ripening while the lycopene content significantly increased in the outer pericarp and columella of the tomatoes at the red ripe stage, as compared to the mature green stage. This observation for the sugar and lycopene signal intensities was further confirmed by HPLC analyses. The non-destructive CSI technique has potential as an efficient tool for the physiological analysis of fruit and vegetables.     

番茄红素的研究概况

综述了番茄红素在国内外的研究动态,介绍了番茄红素的分布、理化性质、化学结构,以及作用机制和生理功能,阐述了番茄红素的应用前景。     

Comparative fine mapping of fruit quality QTLs on chromosome 4 introgressions derived from two wild tomato species

Despite their unsuitability for agricultural production, the wild relatives of crop species represent a largely untapped resource of novel QTLs potentially useful for crop plant improvement. In this regard, previous introgression studies, involving several different wild tomato species, have shown that the long arm of chromosome 4 contains QTLs for many horticulturally important traits including soluble solids content, fruit shape, lycopene content and biochemical composition. However, these earlier studies were unable to determine how many genes control these traits and whether genes affecting the same character from different wild species are allelic or not. In an effort to shed light on these issues,we have constructed a series of lines containing small, overlapping introgressions for portions of the long arm of chromosome 4 from L. peruvianum and L. hirsutum and tested these lines in replicated field trials. The results provide evidence for multiple, non-allelic loci controlling soluble solids and fruit weight. They also show that the loci controlling some traits (e.g. fruit shape, fruit weight, epidermal reticulation) co-localize to the same portions of chromosome 4, a result that maybe attributed to pleiotropy and/or gene dense areas with lower than average recombination. The implications of these finding for molecular breeding and utilization of exotic germplasm are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Heritabilities and Minimum Gene Number Estimates of Carrot Carotenoids

Summary Broad sense heritabilities and gene numbers were estimated for the production of total carotenoids and the major component carotenoids of carrot storage roots: phytoene, ζ-carotene, β-carotene, α-carotene, and lycopene. Two crosses with different backgrounds were evaluated: orange B493 × white QAL and orange Brasilia × dark orange HCM. The HCM (high carotene mass selection), Brasilia and B493 parents had both α-carotene and β-carotene, but HCM had proportionally more α-carotene. Carotene content in F ₂ populations ranged from 522 ppm to 1714 ppm in Brasilia × HCM and from 0 to 695 ppm in B493 × QAL progeny. F ₂ plants segregating for absence of α-carotene were identified in B493 × QAL. Broad-sense heritabilities ranged from 28% to 48% for all carotenes except lycopene and phytoene where estimates were 44% to 89% in the Brasilia × HCM cross, All heritability values exceeded 88% for the B493 × QAL cross, except one estimate for lycopene. The estimated number of genes was 4 conditioning α-carotene, 2 to 3 each for β-carotene and total carotenes and one each for ζ-carotene, lycopene and phytoene in the orange × dark orange cross. In the orange × white cross, the estimates were 4 genes for α-carotene, 1 to 2 each for lycopene and total carotenes and 1 for each of the other carotenes. These results are in general agreement with QTL studies and they provided evidence for continuous inheritance of α-carotene, β-carotene and total carotenoids in the orange × dark orange cross and discrete inheritance for β-carotene and total carotenoids in the orange × white cross.Part of thesis “Biometrical studies and quantitative trait loci associated with major products of the carotenoids pathway of carrot (Daucus carota L.)” presented by the first author as partial fulfillment of the requirement of the PhD degree in Plant Breeding and Plant Genetics, UW-Madison, USA, 2001.     

不同冻存液对奶牛乳腺上皮细胞冻存质量的影响

本试验旨在探究奶牛乳腺上皮细胞（bovine mammary epithelial cells,BMECs）最佳的冻存液以改善乳腺上皮细胞的冻存质量。BMECs传至第5代后分别加入以下10种不同配方的冻存液。A组：85%DMEM+10%胎牛血清+5%DMSO;B组：80%DMEM+10%胎牛血清+10%DMSO;C组：75%DMEM+10%胎牛血清+15%DMSO;D组：70%DMEM+10%胎牛血清+20%DMSO;E组：85%DMEM+5%胎牛血清+10%DMSO;F组：75%DMEM+15%胎牛血清+10%DMSO;G组：70%DMEM+20%胎牛血清+10%DMSO;H组：80%DMEM+10%胎牛血清+10%甘油;I组：70%DMEM+10%胎牛血清+20%甘油;J组：60%DMEM+10%胎牛血清+30%甘油,冻存前统一调整细胞密度到1×106 /mL冻存。分别对复苏后的细胞进行台盼蓝染色计算存活率和PI/Hoechst33258双染计算凋亡率。结果表明：BMECs经不同冻存剂冻存复苏后,细胞活力、形态学及凋亡率表现有所不同,其中B组和G组的活力和24 h贴壁率较其他组高,二者的凋亡率较低,二者之间差异无显著性(P>0.05);传代后B组细胞的生长状况最好。