ABA信号通路对葡萄果皮花青苷生物合成的调控机制研究

以‘红巴拉多’葡萄为试验材料,在转色前期(约花后6周)用300 mg/L的ABA对果穗进行处理,以清水处理为对照;测定不同发育时期葡萄果实的单果重、可滴定酸、可溶性固性物等生理指标,同时测定果皮中总花青苷及ABA含量;检测不同发育时期果皮中ABA信号通路和花青苷生物合成相关基因表达量,克隆6个与花青苷生物合成相关基因的启动子,并预测启动子中的顺式作用元件,在转录调控水平上探讨ABA信号通路对葡萄果皮花青苷生物合成的调控作用。结果表明:(1)ABA处理的葡萄果实可溶性固形物含量明显提高、可滴定酸含量下降。(2)ABA处理显著提高了‘红巴拉多’葡萄果皮的着色水平以及总花青苷和ABA含量。(3)ABA处理后,9个ABA信号通路基因以及6个花青苷生物合成相关基因表达水平明显提高。(4)6个花青苷生物合成相关基因的启动子序列中均含有多个与ABA响应相关的ABRE作用元件。研究发现,9个ABA信号通路基因可能在葡萄果皮着色中发挥着重要作用,其中2个VvABFs转录因子可能直接作用于含有ABRE元件的花青苷生物合成相关基因的启动子序列,推测可通过调控这些基因的转录水平来调控葡萄果皮花青苷的积累。     

红肉葡萄果实发育过程中花青苷组分变化与基因表达规律分析

以红肉葡萄新种质‘钟山红玉’为实验材料,通过高效液相色谱-质谱联用(HPLC-MS)检测‘钟山红玉’从幼果到成熟期果皮、果肉中的花青苷组分及含量,并利用实时荧光定量PCR检测不同发育时期花青苷合成相关基因的表达水平,研究其不同果实发育时期果皮、果肉中的花青苷组分变化,以及相关基因表达规律,探索红肉葡萄果实呈色机制,为葡萄果实品质改良育种提供理论依据。结果表明:(1)‘钟山红玉’果皮和果肉中共检测出13种花青苷且都为单糖苷花青苷,与欧亚种葡萄亲缘关系较近。(2)‘钟山红玉’果皮、果肉中均检测出了欧亚种葡萄中很少有的天竺葵素3-O-葡萄糖苷;果皮中飞燕草素类(飞燕草素-、锦葵色素-、矮牵牛素-)花青苷衍生物含量显著高于矢车菊素类(矢车菊素-、芍药素-)花青苷衍生物,而在果肉中则两大类花青苷含量相近,这与果皮中较高的F3′5′H基因表达量有关,且在果皮中酰基化花青苷比例显著高于果肉。(3)花青苷合成相关基因MYBA2和UFGT在‘钟山红玉’不同发育时期的表达变化规律一致,UFGT基因在果肉中基本不表达,而MYBA2可能是决定‘钟山红玉’果肉转色的关键因子;并且ABA响应相关基因PYL1在‘钟山红玉’发育时期的表达规律与OMT和LDOX基因一致。     

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。     

外源L-谷氨酸对荔枝果实生长与着色以及营养品质的调控作用

以8年生‘妃子笑’荔枝为试验材料,于盛花后45d果面喷施0、1.10、1.30、1.50、1.70和1.90g/L的L-谷氨酸,研究各浓度L-谷氨酸处理对‘妃子笑’荔枝果实生长、果皮着色和果肉营养品质的影响。结果显示:(1)各浓度L-谷氨酸处理对采收期(处理后20d)荔枝果皮中的叶绿素含量无显著影响,但1.30~1.90g/L L-谷氨酸处理采收期荔枝果皮中花青苷含量比对照显著增加92%以上;在采收期,1.10~1.30g/L L-谷氨酸处理的果面色泽参数a*值显著高于对照,色度角h°值、亮度L*、色彩饱和度C值均显著低于对照。(2)与对照相比,1.10g/L L-谷氨酸处理使采收期荔枝果实单果重、纵径和横径分别显著增加30%、4%和6%,而1.90g/L L-谷氨酸处理的单果重、纵径和横径分别比对照降低了1%~2%。(3)L-谷氨酸(1.10、1.30和1.70g/L)处理使荔枝果肉可溶性固形物含量峰值的出现时间比对照延迟5d,缩短了果皮与果肉的发育间隔时期;各浓度L-谷氨酸处理对果肉可滴定酸含量无显著影响,但1.30g/L L-谷氨酸处理使荔枝果肉中维生素C含量比对照提高15%。研究表明,适宜浓度的外源L-谷氨酸处理可显著促进‘妃子笑’荔枝果实生长和果皮着色,并有效改善果肉营养品质,其中以1.30g/L L-谷氨酸处理的综合效果最好,过高浓度L-谷氨酸(1.90g/L)则抑制荔枝生长。     

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。     

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。     

外源ABA和GA3对红肉脐橙果皮主要色素含量变化和果实着色的影响

测定了红肉脐橙(Citrus sinensis Osbesk cv.Cara Cara)果实发育期间和果实转色前用不同浓度的外源ABA和GA3处理后果皮叶绿素和类胡萝卜素的动态含量,并测定了外源ABA和GA3处理后成熟红肉脐橙果皮色泽的表现.结果表明红肉脐橙果皮叶绿素含量于9月20日出现最大值,为0.146 9 mg*g-1FW;类胡萝卜素含量于12月20日达到最大值,为0.032 1 mg*g-1FW;果实转色前,用外源ABA处理后加速了果皮叶绿素的降解,但也抑制了果皮类胡萝卜素的积累,用GA3处理后延缓了果皮叶绿素的降解,同样抑制了果皮类胡萝卜素的积累,严重阻碍了果皮类胡萝卜素的合成;外源ABA或GA3处理均不利于果实色泽品质的提高.     

苹果新品种‘瑞阳’果实主要特性研究

该研究以红色晚熟苹果新品种‘瑞阳’及其母本‘秦冠’、父本‘富士’为试验材料,分析各品种果实发育过程中的生长动态、色泽变化以及采收期对其果实品质的影响,为品种栽培管理和推广应用提供参考。结果表明:(1)在果实生长发育期,‘瑞阳’单果质量的变化与双亲接近,单果质量的日增长高峰出现在花后105d,果实发育前期纵径增长较大,果形指数大,在发育后期果形指数降低,至成熟时果形指数达到0.86,介于父母本‘秦冠’和‘富士’之间。(2)套袋处理使果实着色期的色泽参数a*值和花青苷含量上升,但品种间存在差异,套袋处理对‘瑞阳’的色泽参数a*值和花青苷含量影响不大。(3)随果实采后天数的延长,各采收期‘瑞阳’果实淀粉指数逐渐上升,硬度和可滴定酸逐渐下降,而可溶性固形物含量先上升后下降;‘瑞阳’果实在花后174d采收时,果实的硬度和可滴定酸下降均较少,且果实可溶性固形物含量保持在较高水平,能较好地维持该品种的果实品质。研究发现,‘瑞阳’苹果果实膨大期出现在花后105d前后,果实套袋对其表面色泽和花青苷含量影响不大,在陕西渭北以花后174d前后采收为宜。     

外源ABA和GA3对红肉脐橙果皮色素含量的影响

测定红肉脐橙果实发育期间和果实转色期间施用外源ABA和GA3的果皮中叶绿素和总类胡萝卜素含量变化的结果表明:红肉脐橙果皮中叶绿素总量于9月20日出现最大值,为0.1469 mg·g-1(FW);类胡萝卜素总量于12月21日达到最大值,为0.0321 mg·g-1(FW);转色期用外源ABA处理后果皮叶绿素降解加速,而类胡萝卜素积累受抑;转色期施用GA3并不能延缓果皮叶绿素的降解,但能抑制类胡萝卜素的积累,因而阻碍了果皮类胡萝卜素的合成.     

外源腐胺促进苹果果皮花青苷积累的效应

为了探讨外源施加腐胺对苹果果皮花青苷合成相关基因的调控效应和果实着色的影响, 摘袋当天对苹果品种红富士(Malus domestica Borkh. ‘Red Fuji’)果实喷施50 mg.L-1腐胺(putrescine, Put), 利用分光光度计和高效液相色谱仪分别对苹果果皮花青苷含量及其组成进行了分析; 利用实时荧光定量PCR法检测了转录调节因子MYB1和5个花青苷合成结构基因的转录水平。结果表明: (1) 外源喷施Put对于苹果果皮中花青苷的积累具有明显的促进效应, 在果实采收时, 处理组果皮中的花青苷含量为对照组的1.9倍; (2) 处理果实的果皮中含有矢车菊素阿拉伯糖苷(cyaniding-3-arabinoside, Cy-3-ara), 而在相同条件下, 对照组中未能检测到Cy-3-ara; (3) Put处理对于转录调节因子MYB1和类黄酮3, 5-糖苷转移酶(UDP-glycose: flavonoid 3-O-glycosyltransferase, UFGT)基因的转录有明显的促进作用, 摘袋后第1天和第3天, Put处理组的MYB1转录水平分别为对照组的1.6和2.0倍, UFGT变化趋势与MYB1类似, 查耳酮异构酶(chalcone isomerase, CHI)、花青素苷元还原酶 (anthocyanidin reductase, ANR)和无色花青素加双氧酶(leucoanthocyanidin dioxygenase, LDOX)等基因的转录水平在Put处理初期也表现为明显上升, 特别是 LDOX基因, 其转录水平在处理后第1天和第3天分别达到对照的10.2和3.8倍。在所研究的基因中, 二氢类黄酮还原酶(dihydroflavonol 4-reductase, DFR)基因是唯一一个经Put处理后其转录水平受到强烈抑制的基因, 且这种抑制作用在摘袋后第3天最为明显, 对照组的DFR转录水平为Put处理组的2.3倍。     

Differential expression of anthocyanin biosynthetic genes in relation to anthocyanin accumulation in the pericarp of Litchi chinensis Sonn

Litchi has diverse fruit color phenotypes, yet no research reflects the biochemical background of this diversity. In this study, we evaluated 12 litchi cultivars for chromatic parameters and pigments, and investigated the effects of abscisic acid, forchlorofenron (CPPU), bagging and debagging treatments on fruit coloration in cv. Feizixiao, an unevenly red cultivar. Six genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) were isolated from the pericarp of the fully red litchi cv. Nuomici, and their expression was analyzed in different cultivars and under the above mentioned treatments. Pericarp anthocyanin concentration varied from none to 734 mg m⁻² among the 12 litchi cultivars, which were divided into three coloration types, i.e. non-red (‘Kuixingqingpitian’, ‘Xingqiumili’, ‘Yamulong’and ‘Yongxing No. 2′), unevenly red (‘Feizixiao’ and ‘Sanyuehong’) and fully red (‘Meiguili’, ‘Baila’, Baitangying’ ’Guiwei’, ‘Nuomici’ and ‘Guinuo’). The fully red type cultivars had different levels of anthocyanin but with the same composition. The expression of the six genes, especially LcF3H, LcDFR, LcANS and LcUFGT, in the pericarp of non-red cultivars was much weaker as compared to those red cultivars. Their expression, LcDFR and LcUFGT in particular, was positively correlated with anthocyanin concentrations in the pericarp. These results suggest the late genes in the anthocyanin biosynthetic pathway were coordinately expressed during red coloration of litchi fruits. Low expression of these genes resulted in absence or extremely low anthocyanin accumulation in non-red cultivars. Zero-red pericarp from either immature or CPPU treated fruits appeared to be lacking in anthocyanins due to the absence of UFGT expression. Among these six genes, only the expression of UFGT was found significantly correlated with the pericarp anthocyanin concentration (r = 0.84). These results suggest that UFGT played a predominant role in the anthocyanin accumulation in litchi as well as pericarp coloration of a given cultivar.     

Differential effects of abscisic acid and ethylene on the fruit maturation of <Emphasis Type="Italic">Litchi chinensis</Emphasis> Sonn.

Two litchi cultivars, a well-coloured ‘Nuomici’ and a poorly coloured ‘Feizixiao’, were used to investigate changes in endogenous abscisic acid (ABA) concentration and ethylene production during fruit maturation and to test the effects of exogenous growth regulators on litchi fruit maturation. Abscisic acid concentration in both the aril and pericarp increased with fruit maturation. Transfusion of ABA into the fruit 3 weeks before harvest accelerated, whereas transfusion of 6-benzyl aminopurine (6-BA) retarded sugar accumulation and pigmentation. The effect of 6-BA was assumed to link with the resultant decrease in ABA. In contrast, 1-aminocyclopropane-1-carboxylic acid (ACC) concentration and ACC oxidase (ACO) activities in the aril remained relatively constant during sugar accumulation. Transfusion of aminooxyacetic acid (AOA) significantly decreased ACC concentration but had no effect on sugar accumulation in the aril. These results suggested that endogenous ABA, but not ethylene, was critical for the sugar accumulation. However, the roles of ABA and ethylene in pericarp pigmentation were rather complicated. Application of exogenous ABA promoted anthocyanin synthesis significantly, but had very little effect on chlorophyll degradation. Ethylene production in litchi fruit decreased with development, but a transient increase of endogenous ethylene production was detected just around the colour break in ‘Nuomici’. Enhanced ACO activity in the pericarp was detected during pigmentation. Ethrel at 400 mg l⁻¹ showed no effect on pericarp coloration, but accelerated chlorophyll degradation and anthocyanin synthesis at a much higher concentration (800 mg l⁻¹). Fruit dipped in ABA solution alone yielded no effect on chlorophyll degradation, but the combined use of ABA and Ethrel at 400 mg l⁻¹ enhanced both chlorophyll degradation and anthocyanin synthesis. These results indicated the possible synergistic action of ethylene and ABA during litchi fruit colouration. ABA is suggested to play a more crucial role in anthocyanin synthesis, while ethylene is more important in chlorophyll degradation. ABA can increase the sensitivity of pericarp tissue to ethylene.     

早红考密斯及其芽变果实中花青素含量与相关酶活性变化的研究

以西洋梨早红考密斯及其绿色芽变果实为材料,研究了果实发育期间果皮色泽、花青苷含量及其相关酶活性变化.结果显示:(1)早红考密斯果皮色泽从成熟前的暗红色逐渐变为成熟时的浅红色,并在色泽分布不均匀的地方显出黄色底色,色泽指数(a*)值从花后45 d的16.4降低到成熟时的7.4,降低54.9%;花青苷含量从成熟前的258.4μg?g-1降到成熟时的118.3μg?g-1;早红考密斯果皮色泽和果皮花青苷含量具有密切的相关性.(2)早红考密斯的绿色芽变在果实发育的前期检测不到花青苷,发育后期果实向阳部出现浅红晕,但花青苷含量极低,与亲本差异极显著.(3)果实发育期间,两品种间苯丙氨酸解氨酶(PAL)变化趋势相似,总体呈下降趋势,且早红考密斯的活性总体低于其绿色芽变;两品种查耳酮异构酶(CHI)活性总体变化趋势基本一致,均呈现缓慢上升的趋势,在前期绿色芽变的CHI活性高于其亲本,后期低于亲本;类黄酮3-O-葡(萄)糖基转移酶(UFGT)活性在两品种间的差异较大,在整个果实发育期间早红考密斯的UFGT活性远高于其绿色芽变.研究表明,早红考密斯果皮色泽变化主要由花青苷的含量不同引起;PAL和CHI不是绿色芽变的直接原因;UFGT与花青苷合成密切相关,绿色芽变果皮中UFGT活性显著降低.     

Differential expression of litchi XET genes in relation to fruit growth

Xyloglucan endotransglycosylase (XET) catalyses the transglycosylation of xyloglucan, the major hemicellulose polymer, which has been thought to mediate the cross-linking of cellulose microfibrils in cellular walls and proposed to be involved in the control of cell wall relaxation. To understand the relationship between litchi fruit cracking and gene expression patterns, three XET genes from litchi fruit were identified and then examined for their expression profiles in pericarp and aril tissues at different development stages, using a cracking-resistant cultivar, 'Huaizhi', and a cracking-susceptible cultivar, 'Nuomici'. Three full-length cDNAs of 1267, 1095 and 1156 bp encoding XETs, named LcXET1, LcXET2 and LcXET3, respectively, were isolated from expanding fruit using RT-PCR and RACE-PCR (rapid amplification of cDNA ends) methods. Northern blotting analysis showed that LcXET1 mRNA accumulation occurred much earlier in aril tissues at 59 days after anthesis (DAA) than in pericarp tissues at 73 DAA in 'Nuomici'. However, it appeared at almost the same time (66 DAA) in pericarp and aril tissues in 'Huaizhi', which suggested that differential accumulation of LcXET1 in pericarp and aril tissues in 'Nuomici' and 'Huaizhi' was closely associated with fruit cracking. LcXET2 mRNA accumulation could be detected in pericarp and aril tissues throughout fruit development but exhibited a differential accumulation pattern between pericarp and aril tissues. In the aril of 'Nuomici', intensive signal bands were detectable at 59-73 DAA in rapidly expanding fruits of 'Nuomici' but only weak bands could be found in the pericarp tissues. In contrast, moderate signal bands were detectable both in pericarp and aril tissues of 'Huaizhi' fruits. Furthermore, LcXET3 showed constitutive expression in both pericarp and aril tissues of developing 'Nuomici' and 'Huaizhi' litchi fruit. In addition, differential expression patterns of three XETs genes were observed in different tissues of litchi, with only LcXET1 being fruit-specific. To further address the role of LcXET in fruit cracking, alpha-naphthalene acetic acid (NAA) was used to treat 'Nuomoci' to reduce fruit cracking. Enhanced LcXET1 mRNA accumulation appeared in pericarp while LcXET2 and LcXET3 mRNA accumulation enhanced in aril tissues in the NAA-treated fruits. Thus, LcXET1 is more likely to play a role in reducing litchi fruit cracking than LcXET2 and LcXET3.     

湖南地区塔罗科血橙花青素苷含量研究初报

为了解湖南地区塔罗科血橙Citrus sinensis ‘Tarocco’花青素苷的合成时期和相对含量,通过调查选择合适的采样点,在开花后100 d起每隔30 d采摘果实,利用pH示差法测定塔罗科血橙果肉的花青素苷含量,并以同果园的纽荷尔脐橙C. sinensis ‘Newhall’为对照。结果表明,纽荷尔脐橙在果实发育阶段中不形成花青素,塔罗科血橙的花青素在开花220 d之后开始积累,至250 d时花青素苷含量为5.98 mg?L-1,280 d时花青素苷含量达12.09 mg?L-1。随着塔罗科血橙果实发育,花青素苷含量逐渐增加。     

石榴果色合成相关基因CHS和CHI的表达特性分析

花色苷是类黄酮家族中重要的一类次生代谢产物,对果实呈色起重要作用。CHS (查尔酮合成酶)和CHI (查尔酮异构酶)为花色苷合成提供了前体物质,是花色苷合成所不可或缺的。利用RT-PCR和RACE方法,本研究从石榴果皮中克隆了与花色苷合成相关的CHS基因和CHI基因的cDNA全长,同时采用qRT-PCR研究了这两个基因在三个不同色泽石榴品种‘红宝石’、‘水晶甜’、‘墨石榴’发育期内的表达模式,并分析了果皮花色苷含量变化与基因转录水平的关系。结果表明,石榴中CHS和CHI基因cDNA全长分别为1 197 bp和693 bp,分别编码398和230个氨基酸,命名为PgCHS和PgCHI,在GenBank中的登录号分别为KF841615和KF841616。在氨基酸水平上,Pg CHS与荔枝、葡萄、山竹等果树的同源性达到90%以上。Pg CHI与果树中龙眼、梨、美洲葡萄、桑树等同源性达到70%以上。qRT-PCR结果显示,CHS和CHI基因的表达模式随色泽发育期和品种不同而有差异。在‘红宝石’石榴中,该两个基因都有前期和后期两个表达高峰期;而‘水晶甜’石榴中这两个基因的表达高峰期均出现在中后期;‘墨石榴’发育初期时CHS和CHI的表达量最高,以后的表达量都较低。同一品种内,CHS和CHI的表达具有协同性,两者的协同性表达有利于花色苷及其他类黄酮相关产物的合成。3个品种中CHS和CHI基因的表达与花色苷的积累并不一致。