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‘金华美女’(Camellia japonica ‘Jinhua Meinü’)是山茶(C. japonica L.)名贵品种‘贝拉大玫瑰’(C. japonica Nuccio’s Bella Rossa)的芽变品种。相比绿色叶片的‘贝拉大玫瑰’,‘金华美女’叶芽呈红色,展叶期间的嫩叶呈深紫色,随着叶片生长,深紫色的叶片颜色逐渐淡化并变为红紫色,待叶片成熟后,呈现暗红色,该特性使其具有既可观花也可观叶的特性,提高了其观赏价值和经济价值。
已有研究表明,花青素苷含量变化是影响植物叶片呈红色或者紫色最主要的因素。常见的红叶或紫叶观赏植物,如红叶石楠(Photinia × fraseri ‘Red robin’)、红花檵木(Loropetalum chinense (R. Br.) Oliver var. rubrum Yieh)、紫色酢浆草(Oxalis corniculata L.)、紫叶李(Prunus cerasifera Ehrh.)等植物的叶片中,都含有较多的花青素苷[1-4]。花青素苷为一种类黄酮化合物,其最初是由苯丙氨酸开始,经过一系列复杂的黄酮代谢途径形成花青素,再通过UDP类黄酮糖基转移酶催化,与糖类化合物通过糖苷键形成稳定的花青素苷。目前,花青素苷合成途径中主要的酶以及对应的功能基因在多种植物中均已经研究,在花青素苷合成过程中均发挥重要的作用。在黄洋葱(Allium cepa L.)DFR启动子区域碱基发生突变后,导致该基因无法正常表达,使该洋葱品种表皮从紫色变成黄色[5]。将百子莲(Agapanthus praecox ssp.)的DFR基因转化到烟草(Nicotiana tabacum L.)中,并在烟草中进行过量表达,会使烟草花色由白色变为红色[6]。在连翘(Forsythia suspense L.)转化体系中,只有同时过量表达DFR和ANS基因才能够改变花色(由白色变红色)[7]。在荔枝(Litchi chinensis Sonn.)和石榴(Punica granatum L.)的果皮从青色变为红色的过程中,可以检测到UFGT基因表达明显上调[8-10]。虽然ANR表达产物不直接催化花青素苷合成,但也有证据显示,该基因的功能与花青素苷合成存在反向调控的关系[11-13]。目前,在模式植物的基因功能水平上已经对花青素苷合成相关基因及部分化合物进行含量分析,但很少有报道能够系统地分析基因表达水平与生物化学水平之间的关系。在经济价值较高的红叶或紫叶观赏植物叶片变色机理的研究中,并没有相关报道分析相关基因与化合物含量变化的关系。
本研究以‘金华美女’叶片为材料,以绿叶山茶‘贝拉大玫瑰’、杜鹃红山茶(C. azalea Wei)和红山茶(C. japonica L.)原种作为参照,分别测定叶片4个不同发育时期的DFR、ANS、UFGT、LAR和ANR基因的表达量,以及多酚合成途径中表儿茶素、花青素苷合成途径中矢车菊素-3-O-葡萄糖苷在叶片中的含量变化,以期从两类合成途径主要产物及相关调控基因的表达量层面揭示红叶品种变化机理,为今后开展分子育种,培育彩叶山茶新品种提供理论依据。
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自2015年6月25日起,取3株4年生、健康、长势一致的‘金华美女’扦插苗,种植于中国林科院亚热带林业研究所苗圃中,于2016年4月23日起待其叶片由叶芽展开后,取顶端第1片叶后,待第2个叶芽叶片展开后10 d摘取,待第3个叶芽叶片展开后20 d摘取,待第4个叶芽叶片展开后30 d摘取,共4个时期,分别标记为Ⅰ、Ⅱ、Ⅲ和Ⅳ。每个时期在3个同品种不同植株上重复操作5次。所有样品采摘后均液氮研磨后-80℃冰箱保存备用。另取生长环境相同,同株数的‘贝拉大玫瑰’、杜鹃红山茶和1株红山茶原种作为平行对照。
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叶片总RNA提取按照试剂盒(RN53-EASYspin Plus试剂盒,北京艾德莱生物科技有限公司)说明书进行。所得到RNA经电泳检测应有明显的两条带,同时经分光光度检测,确保所提取RNA的260/280和260/230均介于1.8~2.0,提取完成后置于-80℃下保存。
按照试剂盒(PrimeScriptTM Ⅱ 1st Strand cDNA Synthesis Kit,Takara)进行cDNA合成并将得到的cDNA储存于-20℃下备用。
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根据NCBI数据库查询已知山茶属(Camellia L.)植物的DFR、ANS、LAR、ANR和UFGT cDNA序列,通过实验室现有的杜鹃红山茶转录组数据进行对比,确定相关基因的保守序列。采用NCBI Primer Designing Tool在线设计软件(https://www.ncbi.nlm.nih.gov/tools/primer-blast/)设计1对扩增片段长度约100~150 bp且具高度特异性的荧光定量引物(表 1),GAPDH基因为内参基因。
表 1 荧光定量引物
Table 1. The primers for real-time PCR
基因
Gene name引物序列
The sequence of primersGeneBank DFR F
DFR RGTTCGCGATCCAGCGAATTT
TCCAGAGCAACCCTCAATGGAY648027.1 ANS F
ANS RGCCAAAAGAAGAGCTGACGG
CCTTCTTCAACGCCTTCCGAJN944577.1 LAR F
LAR RCCATCGGAGTTTGGGCATGA
GGCACCCCACACTCTTCTATCKR045740.1 ANR F
ANR RGCTACACCGGTCAACTTTGC
ACACGTTTAACCGTTCCTGCAY641729.1 UFGT F
UFGT RTAACCCTTGGGCTAATCCGC
TGTTGTCCGGGATTGGTGAGKP682360.1 使用试剂盒(SYBR® Premix Ex TaqTM,Takara)和QuantStudioTM 7 Flex荧光定量PCR仪(Applied Biosystems)进行荧光定量PCR实验;采用QuantStudioTM Real-Time PCR Software软件进行数据采集;采用Excel 2010软件进行数据分析。
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叶片总多酚提取参考Inomata等[14]的方法。分别将儿茶素和表儿茶素标准品(购于ChromaDex公司)用甲醇配置成125、62.5、50、25 μg·mL-1 4个浓度的混合标品溶液。
色谱流动相配置及洗脱方法参考Punyasiri等[15]的方法。
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采用Kerio等[16]的方法进行总花青素苷的提取。分别将矢车菊素-葡萄糖苷标准品配置100、50、25、12.5 μg·mL-14个浓度,使用岛津LC-20液相色谱系统(SPD-20A紫外检测器;Inertsustain C18色谱柱:150 mm×46 mm,5 μm)测定标准品溶液标准曲线和样品的矢车菊素-葡萄糖苷浓度,具体色谱流动相配置及洗脱方法参考Li等[17]的方法。
红叶山茶品种花青素苷相关基因表达水平及代谢产物分析
Analysis of Gene Expressing and Metabolites for Synthesising Anthocyanins Influencing the Red Color of Leaves of Camellia Cultivar
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摘要:
目的 通过对花青素苷相关合成基因的表达水平和代谢产物的分析,系统地阐明‘金华美女’叶色变异与基因表达的关系。 方法 以‘金华美女’为材料,‘贝拉大玫瑰’、杜鹃红山茶和红山茶为参照组,使用NCBI Primer Designing Tool设计山茶DFR、ANS、LAR、ANR和UFGT基因的荧光定量引物,使用实时荧光定量PCR仪测定这5个基因在叶片4个发育时期的表达量;采用高效液相色谱仪测定对应时期的多酚合成途径的主要次生代谢产物(儿茶素、表儿茶素),以及花青素苷合成途径的主要代谢产物矢车菊素-3-O-葡萄糖苷的含量。 结果 表明:(1)在4个时期中,红叶品种叶片中DFR基因表达量与对照组差异不显著,ANS、LAR、ANR和UFGT基因在4个时期的表达量与对照组存在显著差异,这说明‘金华美女’类黄酮代谢途径相关基因在表达水平上发生了改变;(2)‘金华美女’叶片中多酚含量明显低于对照组,其中,表儿茶素含量仅为0.04~0.21 mg·g-1,这说明在生理水平上,‘金华美女’叶片中多酚合成途径可能受阻;(3)‘金华美女’叶片中矢车菊素-3-O-葡萄糖苷含量达1.2~1.4 mg·g-1,4个生长阶段均显著高于对照组,这说明‘金华美女’叶片具备持续合成花青素苷的能力。 结论 根据试验结果,推断‘金华美女’叶色变异的主要原因可能是由于花青素还原酶基因的表达水平受到了抑制,降低了矢车菊素催化成表儿茶素的效率,使叶片类黄酮代谢途径中以多酚为主的合成方向转向花青素苷合成方向。 Abstract:Objective By analyzing the quantitative change of structural genes and metabolite of anthocyanin in 4 growth stages, the relationship between leaf-color mutation and gene expression was systematically illustrated. Method The leaves of Camellia japonica 'Jinhua Meinü' were selected taking the leaves of C. japonica 'Nuccio's Bella Rossa', C. azalea, and C. japonica as control. NCBI Primer Designing Tool was used to design the primers and the gene expressing of DFR, ANS, UFGT, ANR and LAR were analyzed by real-time PCR and measured the contents of catechins, epicatechins and cyanidin 3-O-glucoside by HPLC. Result (1) In the four growth stages, no significant difference was found in the expressing of DFR, nevertheless, the expressing of ANS, UFGT, ANR and LAR between red and green cultivar were obviously different, which indicating the express levels of those genes of flavonoids metabolic pathways had been altered in the leaves of C. japonica 'Jinhua Meinü'. (2) The content of polyphenols in the leaves of C. japonica 'Jinhua Meinü' were at a significantly lower level than the control groups, in particular, the content of epicatechins was only 0.04-0.21 mg·g-1, which suggesting the synthetic route of polyphenols were astricted in physiological level. (3) The average content of cyanidin 3-O-glucoside in the leaves of C. japonica 'Jinhua Meinü' was 1.2-1.4 mg·g-1, which was at a significantly higher level than the control groups in all the four growth stages. The content of cyanidin 3-O-glucoside could explain the capacity of synthesizing anthocyanins was kept in the leaves of the red cultivar. Conclusion According to above data, it can be preliminary inferred that the leaf color of C. japonica 'Jinhua Meinü' related to the change of anthocyanins metabolic pathway. -
Key words:
- camellia
- / anthocyanins
- / polyphenols
- / synthetic route
- / gene expressing
- / leaf color
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表 1 荧光定量引物
Table 1. The primers for real-time PCR
基因
Gene name引物序列
The sequence of primersGeneBank DFR F
DFR RGTTCGCGATCCAGCGAATTT
TCCAGAGCAACCCTCAATGGAY648027.1 ANS F
ANS RGCCAAAAGAAGAGCTGACGG
CCTTCTTCAACGCCTTCCGAJN944577.1 LAR F
LAR RCCATCGGAGTTTGGGCATGA
GGCACCCCACACTCTTCTATCKR045740.1 ANR F
ANR RGCTACACCGGTCAACTTTGC
ACACGTTTAACCGTTCCTGCAY641729.1 UFGT F
UFGT RTAACCCTTGGGCTAATCCGC
TGTTGTCCGGGATTGGTGAGKP682360.1 -
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