穗醋栗MYB10基因克隆结构分析及功能验证

为探究花色苷合成相关转录因子MYB10在不同颜色穗醋栗果实着色差异的分子机理,通过cDNA末端快速扩增技术(rapid amplification of cDNA ends, RACE)法从果实花青素含量有较大差异的黑穗醋栗(Ribes nigrum L.)、红穗醋栗(Ribes rubrum L.)和白穗醋栗(Ribes album L.)中分别克隆出MYB10基因,分别命名为RnMYB10 (KY786107)、RrMYB10 (KY786108)和RaMYB10(MW660848)。系统发育分析表明,RnMYB10和RrMYB10在进化上具有同源性。实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)结果表明:黑穗醋栗各时期果实中MYB10表达量均高于红穗醋栗且远远高于白穗醋栗。随着果实直径加大颜色加深,RnMYB10和RrMYB10表达量呈现先上升后下降的趋势(在果实转色程度75%时达到最大值),RaMYB10表达量极低,几乎无表达。过表达RnMYB10和RrMYB10的拟南芥呈现紫色叶柄和叶片,过表达RaMYB10的拟南芥无明显变化。说明...     

2个石榴品种果皮花色苷合成相关基因表达分析

以2个不同红色石榴品种‘红宝石’和‘墨石榴’为试验材料,采用荧光定量PCR方法,分析花色苷合成相关基因CHS、CHI、F3H、DFR、ANS、UFGT等6个基因在果实发育过程中的转录表达特性,同时分析基因表达量与果皮花色苷积累的关系。结果表明:(1)在整个果实发育期内‘墨石榴’花色苷含量明显高于‘红宝石’;随着果实的发育,‘红宝石’果皮中总花色苷含量不断增加,而‘墨石榴’中总花色苷含量初期很高,随后迅速下降,后期维持在较低水平。(2)‘红宝石’中CHS、CHI、F3H、DFR、UFGT等5个基因均在果实发育的早期和晚期出现2个表达高峰,而ANS基因的表达量在整个果实发育期内不断升高;在‘墨石榴’中CHS、CHI、F3H、DFR、ANS等5个基因的表达高峰均出现在早期,随着果实的发育表达量均呈下降变化趋势,但UFGT基因在中期时表达量最高。(3)‘红宝石’石榴的ANS基因表达量与总花色苷含量呈显著正相关,‘墨石榴’中CHS和ANS基因的表达水平与总花色苷含量显著相关。研究认为,花色苷合成相关基因的初期和末期表达差异是2个石榴品种着色差异的主要原因,ANS在‘红宝石’着色中起关键作用,CHS和ANS可能在‘墨石榴’花色苷积累中起重要作用。     

‘脆红李’果实发育过程中花色苷含量及相关基因表达分析

花色苷是一类重要的色素,对李红色的形成必不可少。本研究以‘脆红李’为试材,研究了果实发育过程中叶绿素含量、总花色苷含量及果皮主要花色苷组分和含量的变化规律,并分析了Ps PAL、Ps CHS、Ps CHI、Ps F3H、Ps DFR、Ps ANS和Ps UFGT基因在果实不同发育阶段的表达规律。结果表明,随着‘脆红李’果实的生长发育,果皮和果肉中总叶绿素含量呈逐渐下降的趋势;‘脆红李’果肉中不含花色苷,果皮中的花色苷在转色期才开始积累,成熟时达到最大值,为404.37μg/(g·FW),并以矢车菊素-3-O-葡萄糖苷和矢车菊素-3-O-芸香糖苷为主;花色苷合成相关基因在‘脆红李’不同生长发育时期的果皮和果肉中有着特异性的表达,但只有Ps PAL和Ps UFGT基因的转录水平与花色苷含量的正相关性达到极显著水平,表明这两个基因对‘脆红李’果实的着色有着异常重要的调控作用。     

黑穗醋栗红色素的研究

黑穗醋栗(Ribes nigrum L.)别名黑豆、黑加伦、斯马劳金、紫梅等,为虎耳草科茶藨子属(Ribs)的多年生小灌木,果实成熟后呈黑紫色浆果。原产于欧洲,我国栽培的黑穗醋栗系十月革命后由俄侨引入,主要栽培于东北三省,以黑龙江和吉林省面积最大。当前,黑穗醋栗果实主要用于酿造高级果酒如黑加伦酒、黑加伦香槟酒以及汽水等。由于栽培面积迅速发展,产量越来越大,只靠造酒是用不完的,有过剩的趋势。黑穗醋栗果实营养丰富,含有大量红色素。为了充分利用这一资源,我们进行了色素的提取和性质试验,现将结果报告如下。     

不同栽培区域和年份的红皮砂梨果皮着色特性

该研究以红皮砂梨品种‘八月红’和‘红香酥’为试材,比较了不同栽培地区和年份的红皮砂梨着色规律、花色素苷组分及含量,以及相关代谢酶的变化。结果显示:（1）‘八月红’和‘红香酥’果皮花色素苷含量变化规律由其品种特性决定,同一品种的果皮花色素苷绝对含量受不同栽培区域和年份的影响,其组成和含量变化趋势不受影响;但不同品种的花色素苷含量变化以及着色规律有所不同。（2）红色砂梨果皮着色主要与矢车菊-3-半乳糖苷含量变化一致,且随着梨果实的发育和着色加深,果实中UDP-葡萄糖:类黄酮-3-O葡萄糖苷转移酶（UFGT）、二氢黄酮醇-4-还原酶（DFR）活性呈上升趋势,而苯丙氨酸解氨酶(PAL)活性随着果实发育而下降。（3）相关性分析表明,两品种梨果实花色苷含量与其UFGT、DFR活性呈显著或极显著正相关关系,而与PAL活性相关性不一致。研究表明,梨果皮中的UFGT、DFR是影响红皮砂梨着色的重要酶。     

果袋类型对‘紫金红霞’葡萄果实品质及花色苷合成相关基因表达的影响

以‘紫金红霞’葡萄为试验材料,在果实转色前分别进行5种不同类型果袋(白色纸袋、无纺布 白纸双层袋、绿色纸袋、蓝色纸袋、棕色纸袋)套袋处理,以不套袋为对照,测定不同发育时期葡萄果实的单果重、果粒纵横径、可溶性固形物、可滴定酸,及总花色苷含量等生理指标,并利用qRT PCR技术分析不同发育时期花色苷合成相关基因的表达水平,探索不同类型果袋对‘紫金红霞’葡萄果实品质、花色苷含量及花色苷合成相关基因表达的影响。结果表明：(1)白色、蓝色和棕色果袋不利于成熟果实中可溶性固形物含量的升高,蓝色和棕色果袋不利于成熟果实中可滴定酸含量的降低。(2)套袋处理会使果实色泽指数显著降低,除无纺布 白纸双层袋未显著降低成熟期果皮中花色苷含量外,其他套袋处理均显著降低了果皮中总花色苷含量。(3)套袋处理对6个花色苷合成相关基因的表达主要表现为抑制作用,但无纺布 白纸双层袋对成熟期F3′H和UFGT基因的表达、白色纸袋对成熟期MYBA1、DFR和LDOX基因的表达则具有促进作用。研究发现,无纺布 白纸双层袋对成熟期果实的内在品质和果皮着色影响最小,可用于‘紫金红霞’果实套袋,其次为白色和绿色纸袋;蓝色和棕色纸袋可使葡萄果实的品质大幅降低,故不可用于实际生产中葡萄果实的套袋。     

葡萄果实成熟期花色苷与白藜芦醇合成相关基因的表达

以酿酒葡萄‘赤霞珠’为试材,采用pH示差法和高效液相色谱(HPLC)法分别测定葡萄成熟期果皮花色苷和白藜芦醇含量,用实时荧光定量PCR检测两者合成途径中相关基因的表达量,分析花色苷含量和白藜芦醇含量与其相关基因表达的关系,以揭示结构基因与调控基因的调控机制,为筛选富含花色苷和白藜芦醇的酿酒葡萄提供理论依据。结果显示:(1)葡萄果皮花色苷含量在花后112d达到最高值(0.77mg/g),反式白藜芦醇含量在花后126d达到最高值(30.87μg/g)。(2)花色苷和白藜芦醇合成途径中,CHSs、CHI、STS、UFGT、MybA1、MybA2基因的表达量除花后98d下调外,其余时间均呈上调表达,而Myb5a则始终呈上调表达。(3)相关分析表明,STS基因表达量与CHS1、CHS2基因表达量呈极显著和显著正相关关系,MybA1、MybA2基因表达量与CHSs、CHI、STS、UFGT基因的表达量呈极显著正相关关系;Myb5a基因表达量与CHS3基因表达量呈极显著正相关关系。研究表明,部分结构基因的表达与花色苷和白藜芦醇的变化不同步,MybA1和MybA2可能调控花色苷合成途径中多个结构基因的表达,花色苷与白藜芦醇的关系并不固定,而是处在动态变化中。     

桃树体不同部位果实着色差异及其与环境因子的关系研究

为探讨树体冠层不同部位桃果实着色机制差异及其与环境因子的关系,以晚熟桃品种‘霞晖8号’为试材,研究了果实3个典型发育时期(硬核期、膨大期、成熟期)树体冠层上部、中部外围、中部内膛和下部的温度、光照环境因子变化动态,并就其对果皮色泽、色素含量的影响及与果实着色相关的基因表达特点进行解析。结果表明:(1)与冠层下部果实相比,‘霞晖8号’冠层上部、中部外围和中部内膛成熟果实果皮a~*/b~*(红色饱和度/黄色饱和度)显著较高。(2)冠层下部成熟果实的果皮花色素苷含量最低而叶绿素含量最高,且与其他部位间差异显著。(3)果实不同发育时期花色素苷合成相关基因的表达量差异表明,果皮花色素苷合成是多基因协同调控的过程。(4)果实转色前,低光照强度抑制了花色素苷合成相关基因的表达,其中对UFGT、DFR、CHS基因的调控作用最明显;果实成熟期,与高光照条件相比,低光照条件下果皮花色素苷合成相关基因上调表达。研究认为,树体冠层不同部位光照条件差异可能是导致果实着色差异的主要环境因素之一,它通过调节与果皮花色素苷积累相关的基因表达水平控制果皮的着色。     

穗醛栗叶片中黄酮类物质的研究

本试验以小浆果类果树穗醋栗（黑、红、白）的叶片为试材,利用分光光度法。测定了三种穗醋栗叶（鲜样、干样）中总黄酮的含量及不同提取次数下总黄酮的浸提率,确定了最佳提取次数。结果表明：白穗醋栗叶片中总黄酮含量最高,鲜样785．10mg/100g,干样189.01mg/100g;红穗醋栗叶片次之,鲜样393.22mg／100g,干样1597.73mg／100g;黑穗醋栗叶片中总黄酮含量相对较少,鲜样151.59mg/100g,干样265.03mg/100g。三种穗醋栗叶片浸提三次,总黄酮的浸提率可达到97％以上。此外,利用定性试验（颜色反应）并和标准品芦丁的试验做比较,初步确定三种穗醋栗叶片所含的黄酮类化合物主要是黄酮和黄酮醇两类。     

石榴PgUGT基因表达特性与重组表达分析北大核心CSCD

由糖基转移酶参与的糖基化反应是植物次生代谢产物合成中最广泛的一种修饰方式,也是次生代谢产物结构多样性的机制之一。该研究基于石榴(Punica granatum L.)转录组数据,以石榴果皮为材料,采用RT-PCR克隆得到石榴UDP-糖基转移酶(UDP-glycosyltransferase,UGT)基因(PgUGT);采用生物信息学技术对编码蛋白的基本特性进行分析,并构建系统发育树;采用实时荧光定量PCR(qRT-PCR)分析该基因在果实发育期内的表达模式;结合果实发育期内的总类黄酮和总花色苷含量,分析了基因表达与总类黄酮和总花色苷合成的关系;构建PgUGT的原核表达载体,对其进行原核重组表达。结果表明:(1)成功克隆得到石榴PgUGT基因(GenBank登录号为MW414607);PgUGT基因具有一个1557 bp的开放阅读框(ORF),编码518个氨基酸,预测蛋白分子质量为55.9 kD,等电点为6.55,为不稳定的亲水蛋白;PgUGT具有糖基转移酶家族保守的PSPG基序,属于GT-B糖基转移酶基因家族。进化树分析表明,该编码蛋白属于拟南芥UGT的F类群,与葡萄和草莓的UGT亲缘关系较近。(2)qRT-PCR结果表明,石榴PgUGT基因的表达量在果实发育期内呈先升后降的模式,与总类黄酮含量的逐渐下降和总花色苷含量的逐渐上升趋势并不完全一致。(3)成功构建原核表达载体pCZN1-PgUGT;重组原核表达结果显示,重组质粒在大肠杆菌中为可溶性表达,表达的蛋白分子量约为58 kD。该研究结果为PgUGT基因在石榴类黄酮糖基化反应中的作用及功能奠定了基础。     

Expression of the grape dihydroflavonol reductase gene and analysis of its promoter region

Dihydroflavonol reductase (DFR) is a key enzyme involved in anthocyanin biosynthesis and proanthocyanidin synthesis in grape. DFR catalyses the reduction of dihydroflavonols to leucoanthocyanidins in the anthocyanin pathway. The DFR products, the leucoanthocyanidins, are substrates for the next step in the anthocyanin pathway and are also the substrates for the proanthocyanidin pathway. In the present study the promoter of the grape dfr gene was cloned. Analysis of the dfr promoter sequence revealed the existence of several putative DNA binding motifs. The dfr promoter was fused to the uidA gene and the control of this fusion and the endogenous dfr gene expression, was studied in transformed plants and in red cell suspension originated from fruits. The dfr promoter-uidA gene fusion was expressed in leaves, roots and stems. Deletions of the dfr promoter influenced the specificity of the expression of the GUS gene fusion in plantlet roots and the level of expression in plants and in the red cell suspension originated from fruits. The deletion analysis of the dfr promoter suggests that a specific sequence located between -725 to -233 might be involved in expression of the dfr gene in fruits. Light, calcium and sucrose induced the dfr gene expression. In the transformed suspension cultures, expression of both the endogenous dfr gene and the dfr promoter-uidA gene fusions was induced by white light. The induction by both light and calcium suggests the possible involvement of a UV receptors signal transduction pathway in the induction of the dfr gene. The induction of the dfr gene and the dfr promoter-uidA gene fusions by light and sucrose indicates a close interaction between sucrose and light signalling pathways.     

不同花色黄芩中dfr基因的克隆及时空表达分析

二氢黄酮醇-4-还原酶(Dihydroflavonol-4-reductase,Dfr)是类黄酮生物合成途径中调控花青素和原花青素合成的关键酶。为探究dfr基因在同一生态环境下不同花色黄芩中的差异,以白色、紫红色和紫色3种花色的黄芩花瓣cDNA为模板,基于同源克隆和RACE技术从中克隆得到3个完整的dfr基因全长序列,分别命名为Sbdfr1、Sbdfr2、Sbdfr3,并采用生物信息学软件对其相关结构进行分析。结果表明,3个Dfr蛋白均存在高度保守的NADPH结合位点和底物特异性结合位点。系统进化分析表明,三者与已知粘毛黄芩(GenBank登录号:ACV49882.1)聚为一簇,亲缘关系最近;关键结构域和3D模型分析发现3个Dfr蛋白表面催化活性区域均存在差异,其独特的结构特性为研究Dfr底物特异性提供了有利条件。qRT-PCR分析显示,dfr在3种花色黄芩盛花期除根外的其他组织中均有不同程度的表达;在花瓣发育进程中,dfr基因在白花和紫花黄芩中的表达量均呈上升趋势,在紫红花黄芩中的表达量先缓慢上升后下降,之后再迅速上升至最大值。研究结果为进一步深入探究Dfr底物选择性的机理及功能提供一定的理论基础,对阐明黄芩花色差异变化的分子机理具有重要的科学研究价值。     

Anthocyanin-rich black currant (Ribes nigrum L.) extract affords chemoprevention against diethylnitrosamine-induced hepatocellular carcinogenesis in rats

Anthocyanins are known to possess potent anticarcinogenic properties against several cancers thus demonstrating potential for cancer prevention. Black currant (Ribes nigrum L., Grossulariaceae) fruits have a high anthocyanin content. This “superfruit” is known to possess various pharmacological effects including alleviation of chronic oxidative stress and inflammation. In contrast to a large volume of literature on the health benefits of black currant, limited evidence on antitumor effects of black currant exists with virtually no data on the prevention of experimental carcinogenesis. In the current study, we have investigated the chemopreventive effects of an anthocyanin-rich black currant skin extract (BCSE) utilizing our well-characterized model of rat liver carcinogenesis. Initiation of hepatocarcinogenesis was done by intraperitoneal injection of diethylnitrosamine (DENA) followed by promotion with phenobarbital. The rats were exposed to dietary BCSE for 4 weeks prior to initiation, and the treatment was continued for 22 consecutive weeks. BCSE dose-dependently decreased the incidence, total number, multiplicity, size and volume of preneoplastic hepatic nodules. The antihepatocarcinogenic effect of BCSE was confirmed by histopathological examination of liver sections. Immunohistochemical analysis of proliferating cell nuclear antigen and DNA fragmentation revealed BCSE-mediated inhibition of abnormal cell proliferation and induction of apoptosis in DENA-induced rat liver tumorigenesis respectively. Mechanistic studies revealed that BCSE-mediated proapototic signal during experimental hepatocarcinogenesis may be propagated via the up-regulation of Bax and down-regulation of Bcl-2 expression at the translational level. These results along with a safety profile of BCSE encourage the development of black currant bioactive constituents as chemopreventive agents for human liver cancer.     

cDNA cloning and expression pattern of cinnamate-4-hydroxylase in the Korean black raspberry

Cinnamate-4-hydroxylase (C4H) is a key enzyme in the phenylpropanoid pathway, which is responsible for synthesizing a variety of secondary metabolites that participate in development and adaptation. In this study, we isolated a full-length cDNA of the C4H gene from the Korean black raspberry (Rubus sp.) and found that this gene existed as a single gene. By comparing the deduced amino acid sequence of Rubus sp. C4H with other sequences reported previously we determined that this sequence was highly conserved among widely divergent plant species. In addition, quantitative real time PCR studies indicated that the C4H gene had a differential expression pattern during fruit development, where gene expression was first detected in green fruit and was then remarkably reduced in yellow fruit, followed by an increase in red and black fruit. To investigate the two peaks in expression observed during fruit development and ripening, we measured the flavonoid content. The content of the major flavanol of Korean black raspberry fruits was determined to be highest at the beginning of fruit development, followed by a gradually decrease according to the developmental stages. In contrast, the content of anthocyanins during the progress of ripening was dramatically increased. Our results suggest that the C4H gene in Korean black raspberry plays a role during color development at the late stages of fruit ripening, whereas the expression of C4H gene during the early stages may be related to the accumulation of flavanols.     

Detachment-accelerated ripening and senescence of strawberry (Fragaria × ananassa Duch. cv. Akihime) fruit and the regulation role of multiple phytohormones

To study the detachment stress on the ripeness of strawberry fruit, physiological characteristics of strawberry fruit on and off plant during ripeness and senescence processes were investigated. The results indicated that the ripeness of strawberry fruit upon detachment was accelerated, in terms of firmness, soluble solid content and especially color development. The color of fruit off plant changed rapidly from white to full red in 1–2 days. The respiratory rate in fruit off plant was strengthened, higher than that on plant. Abscisic acid level and ethylene production in fruit off plant were also higher than those on plant and auxin degradation was exacerbated by detachment. Expression levels of FaMYB1, FabHLH3 and FaTTG1 were generally reduced with phenotypes of redder color and more anthocyanin accumulation in fruit off plant. Results also suggested that the detachment initially stimulated ethylene and abscisic acid production and auxin degradation, which modulated ripening-related gene expression and at last enhanced fruit pigmentation.     

Effects of anthocyanin and carotenoid combinations on foliage and immature fruit color of Capsicum annuum L

Shades ranging from violet to black pigmentation in pepper (Capsicum annuum L.) are attributed to anthocyanin accumulation. High-performance liquid chromatography and mass spectrometry analysis of violet and black fruit tissue identified a single anthocyanin that was determined to be delphinidin-3-p-coumaroyl-rutinoside-5-glucoside. Leaf tissue of a black-pigmented foliage genotype contained the same anthocyanin found in fruit but at a considerably higher concentration in comparison to violet and black fruit tissue. Fruit chlorophyll concentration was approximately 14-fold higher in black fruit in comparison to violet fruit that contained relatively little chlorophyll. Beta-carotene, lutein, violaxanthin, and neoxanthin carotenoid concentrations in black fruit were also significantly greater in comparison to violet fruit. High concentrations of delphinidin in combination with chlorophyll and accessory carotenoid pigments produced the characteristic black pigmentation observed in fruits and leaves of selected genotypes. Anthocyanins were accumulated in the outer mesocarp of violet and black fruit and in the palisade and mesophyll cells of black leaves. Consistent with chlorophyll content of respective genotypes, chloroplast density was greater in cells of black fruits. Utilizing Capsicum pigment variants, we determine the biochemical factors responsible for violet versus black-pigmented pepper tissue in the context of described pepper color genes.     

A PCR-based diagnostic tool for distinguishing grape skin color mutants

Berry skin color mutants are phenotypically different from their original cultivars, but they show identical molecular profile if analysed by using microsatellite markers. This work gives an easy, inexpensive and quick diagnostic tool to discriminate these somatic variants. We distinguished some grape (Vitis vinifera L.) skin color mutants from white to red or pink and from black to grey, pink or white and we investigated their molecular bases by single-strand conformational polymorphism (SSCP), single base primer extension and coding sequence analysis of anthocyanin biosynthetic enzyme genes and by polymerase chain reaction (PCR) analysis of VvmybA1 regulatory gene. Analyses of structural genes did not reveal polymorphisms between wild type and mutant cultivars but only among different varieties, whereas the study of VvmybA1 regulatory gene has given important outcomes for color mutants characterisation. The discrimination between white wild type and its derived colored mutant and between black wild type and white mutant has been obtained through a simple test of amplification for presence/absence. The discrimination between black wild type and less colored mutant has occurred through a quantitative result on agarose gel confirmed by real-time PCR analysis: the amount of functional allele in less colored somatic variants genome was about one-fourth of the correspondent quantity in original black cultivars genome.