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。     

石榴PgUFGT基因克隆及表达分析

利用RT-PCR和RACE方法,从石榴(Punica granatum L.)果皮中克隆到一个类黄酮糖基转移酶(UFGT)基因(PgUFGT)全长cDNA序列(GenBank登录号为KF841620)。PgUFGT基因编码区1 476bp,编码491个氨基酸。PgUFGT蛋白具有保守PSPG基序、UDP-糖基转移酶家族结构域和UDP-葡萄糖醛酸基/葡萄糖基转移酶保守域(UDPGT),与其他植物UFGT蛋白一致性较高;系统进化树分析结果表明,PgUFGT属于类黄酮3-O-糖基转移酶类。荧光定量qRT-PCR结果表明,PgUFGT基因在‘红宝石’和‘水晶甜’2个石榴品种的发育期内具有不同的表达模式,PgUFGT在‘红宝石’石榴中有2个转录表达高峰,而在‘水晶甜’石榴中仅有1个表达高峰,表明PgUFGT可能在2个石榴品种中具有不同的催化作用。该研究结果为进一步研究石榴果实色泽形成的分子机制奠定了基础。     

不同红梨果皮类黄酮合成基因表达模式分析

采用半定量和荧光定量PCR方法,分析10个类黄酮合成基因在梨品种‘红星’和‘满天红’成熟果皮中的转录特性以及光照对基因表达的影响.结果表明:‘满天红’花色苷合成上游基因(CHS、CHI)的表达量高于‘红星',而下游基因(F3H、DFR、ANS)以及黄酮醇(FLS)和原花色素(LAR、ANR)合成相关基因的表达量却正好相反.套袋去除光照可使所有被检测基因的转录水平降低,F3GT和FLS最明显,表达量差异达20～30倍以上,且套袋‘红星’中PAL、F3H、DFR、ANS、LAR、ANR基因的表达量仍高于不套袋‘满天红’.研究认为,花色苷合成下游基因转录水平的差异是2个红色梨品种间着色不同的主要原因,而F3GT是光照调控‘红星’着色的关键基因.     

桂花查尔酮异构酶OfCHI基因的克隆与表达分析

查尔酮异构酶CHI是花青素苷合成途径中的关键酶。为了解桂花花青苷的合成机理,该研究对3个桂花品种的花青苷含量进行了测定。结果显示:(1)‘橙红丹桂’花中的花青苷含量最高,‘金桂’和‘早银桂’中花青苷含量较低。(2)利用RACE和RT-PCR方法获得了桂花查尔酮异构酶基因(OfCHI)的全长cDNA序列1 069bp,该基因编码248个氨基酸,相对分子量为26.85kD,等电点为6.34。(3)多重序列比对显示,OfCHI与金花茶CnCHI、忍冬LjCHI、石榴PgCHI的相似性分别为68.13%、65.86%和63.53%,氨基酸序列中含有CHI蛋白的活性位点Thr47、Tyr108、Met115以及Ser192;系统进化树分析表明,OfCHI与其他物种起源相同,而与油橄榄OeCHI亲缘关系最近。(4)利用qRT-PCR对不同桂花品种、不同组织中OfCHI的表达量检测结果显示,OfCHI在‘橙红丹桂’花中表达量最高,在‘金桂’和‘早银桂’中表达量较低;OfCHI在‘橙红丹桂’、‘金桂’和‘早银桂’的花、茎、叶中均有表达,且表达趋势相同,均为叶中表达量最高。该研究为揭示桂花青素苷的合成机理奠定了理论基础,并为培育不同花色的桂花新品种提供了基因专利。     

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。     

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。     

‘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。     

不同花色菊花品种花色素结构基因的表达特性

对红色、黄色、粉紫色和白色菊花品种不同开放度的花序舌状花中CHS、CHI、DFR、F3H、F3′H和3GT基因的表达量进行了相对定量分析。结果表显示:6个基因的表达因不同花色、不同发育阶段而异。‘钟山红鹰’(红色)中各基因的表达量均较高,且均在Ⅱ(松蕾期)或Ⅲ(半开期)期达到峰值,其中DFR、3GT基因的表达量远高于其他花色品种。‘金陵娇黄’(黄色)中CHS、CHI基因表达量较高,且Ⅰ(紧蕾期)、Ⅱ期表达量高于Ⅲ、Ⅳ(盛开期)期;3GT、DFR基因表达量分别高或低于‘金陵笑靥’(粉紫色)品种中相应基因的表达量,但均比红色品种低;F3H在4个品种中表达量最低,F3′H表达量接近或略低于红色或粉紫色品种,且各阶段表达水平较稳定。‘金陵笑靥’中DFR表达量仅次于‘钟山红鹰’,3GT和CHS表达量低于红色与黄色品种。‘钟山雪桂’(白色)中各基因仅有微量表达,除F3H外各基因的表达量明显低于其他花色品种。研究表明,花色素结构基因DFR、3GT是菊花花色素合成的关键基因,DFR很可能是限速关键基因,一定表达水平的CHS、CHI也是菊花花色素合成所必须的,F3H基因与花色素合成不存在直接相关。     

花青素生物合成关键酶的研究进展

花青素是植物花呈现不同色彩的物质基础,其生物合成途径主要受到查尔酮合成酶(CHS)、查尔酮异构酶(CHI)、黄烷酮3-羟化酶(F3H)、类黄酮3'-羟化酶(F3'H)、类黄酮3’,5’-羟化酶(F3'5'H)、二羟基黄酮醇还原酶(DFR)、花色素苷合成酶(ANS)以及类黄酮3-O-糖基转移酶(UFGT)等关键酶的控制.主要介绍花青苷生物合成途径、关键酶晶体结构及利用基因工程改造花色的研究进展,讨论目前花色改造存在的问题,并对今后的研究前景进行展望.     

外源脱落酸对桃果实着色及相关基因表达的影响

以‘美香’桃为试验材料,于果实着色前用1 000、500、300mg/L的脱落酸(ABA)溶液处理果实,研究了ABA处理促进桃果皮着色的效果以及对果皮花色素苷合成相关基因表达的影响。结果表明:ABA处理显著提高了成熟果实可溶性固形物含量和果皮花色素苷含量;明显改善了果实着色,且以1 000mg/L效果最为明显。ABA处理显著促进了查尔酮合成酶基因(CHS)和二氢黄酮醇4-还原酶基因(DFR)的前期转录水平,同时促进了类黄酮葡萄糖苷转移酶基因(UFGT)和花色素合成酶基因(LDOX)表达高峰的前移。据此推测ABA可能参与了桃果实花色素苷合成的调控,对花色素苷合成具有促进作用。     

乙烯利处理对葡萄花色苷合成相关基因表达的影响

利用荧光定量PCR技术分析‘京优’葡萄果实成熟过程中,花色苷生物合成途径相关酶基因mRNA转录水平的变化以及乙烯利处理对果皮中花色苷含量和关键酶基因转录水平的影响。结果显示,葡萄果实发育进入着色期,花色苷合成过程中主要相关基因(CHSs、CHIs、F3Hs、F3′H、F3′5′H、DFR、LDOX、UFGT、OMT和GST)和转录因子(MybA1和MybA1-2)转录水平都显著提高,其中UFGT、GST、MybA1和CHSs、CHIs、F3Hs基因家族中的CHS3、CHI2、F3H2随着花色苷合成而大量转录;乙烯利处理能够增强花色苷合成相关基因的转录,使其转录时期前移和转录水平提高,其中对GST、UFGT和MybA1转录的促进作用最明显。相关性分析表明,花色苷合成与一些花色苷合成相关基因(CHS3、CHI2、F3H2、F3′5′H、UFGT、GST)和转录因子(MybA1)的转录水平呈显著或极显著正相关;与CHS1、CHS2、CHI1、F3H1、DFR、F3′H、LDOX和OMT转录水平的相关性均不显著。本研究结果为进一步阐明花色苷生物合成机理和花色苷类色素的生产应用提供一定的理论依据。     

Analysis of the Expression of Anthocyanin Pathway Genes in Developing Vitis vinifera L. cv Shiraz Grape Berries and the Implications for Pathway Regulation

Anthocyanin synthesis in Vitis vinifera L. cv Shiraz grape berries began 10 weeks postflowering and continued throughout berry ripening. Expression of seven genes of the anthocyanin biosynthetic pathway (phenylalanine ammonia lyase [PAL], chalcone synthase [CHS], chalcone isomerase [CHI], flavanone-3-hydroxylase [F3H], dihydroflavonol 4-reductase [DFR], leucoanthocyanidin dioxygen-ase [LDOX], and UDP glucose-flavonoid 3-o-glucosyl transferase [UFGT]) was determined. In flowers and grape berry skins, expression of all of the genes, except UFGT, was detected up to 4 weeks postflowering, followed by a reduction in this expression 6 to 8 weeks postflowering. Expression of CHS, CHI, F3H, DFR, LDOX, and UFGT then increased 10 weeks postflowering, coinciding with the onset of anthocyanin synthesis. In grape berry flesh, no PAL or UFGT expression was detected at any stage of development, but CHS, CHI, F3H, DFR, and LDOX were expressed up to 4 weeks postflowering. These results indicate that the onset of anthocyanin synthesis in ripening grape berry skins coincides with a coordinated increase in expression of a number of genes in the anthocyanin biosynthetic pathway, suggesting the involvement of regulatory genes. UFGT is regulated independently of the other genes, suggesting that in grapes the major control point in this pathway is later than that observed in maize, petunia, and snapdragon.     

Regulation of gene expression involved in flavonol and anthocyanin biosynthesis during petal development in lisianthus (Eustoma grandiflorum)

To elucidate gene regulation of flower colour formation, the gene expressions of the enzymes involved in flavonoid biosynthesis were investigated in correlation with their product during floral development in lisianthus. Full-length cDNA clones of major responsible genes in the central flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3',5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS), were isolated and characterized. In lisianthus, the stage of the accumulation of flavonols and anthocyanins was shown to be divided clearly. The flavonol content increased prior to anthocyanin accumulation during floral development and declined when anthocyanin began to accumulate. CHS, CHI, and F3H were necessary for both flavonol and anthocyanin biosynthesis and were coordinately expressed throughout all stages of floral development; their expressions were activated independently at the stages corresponding to flavonol accumulation and anthocyanin accumulation, respectively. Consistent with flavonol and anthocyanin accumulation patterns, FLS, a key enzyme in flavonol biosynthesis, was expressed prior to the expression of the genes involved in anthocyanin biosynthesis. The genes encoding F3'5'H, DFR, and ANS were expressed at later stages, just before pigmentation. The genes responsible for the flavonoid pathways branching to anthocyanins and flavonols were strictly regulated and were coordinated temporally to correspond to the biosynthetic order of their respective enzymes in the pathways, as well as in specific organs. In lisianthus, FLS and DFR, at the position of branching to flavonols and anthocyanins, were supposed to play a critical role in regulation of each biosynthesis.     

Differential gene expression analysis of ‘Granny Smith’ apple (Malus domestica Borkh.) during fruit skin coloration

‘Granny Smith’ apples growing under normal sunlight develop green skin, whereas the peel turns red due to anthocyanin accumulation after the removal of a bagging treatment. Two anthocyanins, Cyanidin 3-O-galactoside (cy3-gal) and Cyanidin 3-O-arabinoside (cy3-ara), were detected in the red ‘Granny Smith’ apple peels, and cy3-gal was determined to be chiefly responsible for the red color. The content of cy3-gal was more than 98% of the total anthocyanin in the red ‘Granny Smith’ peels. To better understand the molecular basis of anthocyanin biosynthesis in ‘Granny Smith’ apples, we performed a quantitative real-time PCR (qRT-PCR) analysis of anthocyanin biosynthetic genes (MdCHS, MdF3H, MdDFR, MdANS, MdUFGT, and MdMYB1). Our results indicate that the expression of these genes (except MdCHS) was associated with increased anthocyanin accumulation in the skin of ‘Granny Smith’ apples. Four selected genes obtained from the ‘Granny Smith’ skin cDNA library, phytoene synthase (PSY), WD40 repeat protein, polygalacturonase (PG), and galactosidase (GAL), were also confirmed by qRT-PCR. We found that these genes were differently expressed during ‘Granny Smith’ apple skin coloration, suggesting that they are directly or indirectly involved in pigment accumulation. In conclusion, anthocyanin biosynthesis in ‘Granny Smith’ apples is the result of interactions between multiple enzymes in the anthocyanin biosynthesis pathway, and the coloring mechanism of ‘Granny Smith’ apples may be similar to that of red-skinned cultivars.     

Methyl jasmonate affects phenolic metabolism and gene expression in blueberry (Vaccinium corymbosum)

Blueberry (Vaccinium corymbosum) is a fruit very much appreciated by consumers for its antioxidant potential and health‐promoting traits. Its beneficial potential properties are mainly due to a high content of anthocyanins and their amount can change after elicitation with methyl jasmonate. The aim of this work is to evaluate the changes in expression of several genes, accumulation of phenolic compounds and alterations in antioxidant potential in two different blueberry cultivars (‘Duke’ and ‘Blueray’) in response to methyl jasmonate (0.1 mM). Results showed that 9 h after treatment, the expression of phenylalanine ammonium lyase, chalcone synthase and anthocyanidin synthase genes was stimulated more in the ‘Blueray’ variety. Among the phenols measured an increase was recorded also for epicatechin and anthocyanin concentrations. ‘Duke’ is a richer sourche of anthocyanins compared to ‘Blueray’, treatment with methyl jasmonate promoted in ‘Blueray’ an increase in pigments as well as in the antioxidant potential, especially in fully ripe berries, but treated ‘Duke’ berries had greater levels, which were not induced by methyl jasmonate treatment. In conclusion, methyl jasmonate was, in some cases, an effective elicitor of phenolic metabolism and gene expression in blueberry, though with different intensity between cultivars.