Welcome to Chinese Journal of Tropical Crops,

Chinese Journal of Tropical Crops >

2019 , Vol. 40 >Issue 10: 2091 - 2096

DOI: https://doi.org/10.3969/j.issn.1000-2561.2019.10.025

Crop Culture and Nutrition, Genetic Breeding

SmCCoAOMT Positively Regulates the Peel Coloring in Eggplant under High Temperature Stress

  • LYU Lingling 1 ,
  • FENG Xuefeng 2 ,
  • LI Ke 3 ,
  • LI Wei 3
Expand
  • 1. Huaihua University, Huaihua, Hunan 418008, China
  • 2. Shangqiu University, Shangqiu, Henan 476000, China
  • 3. South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences / Hainan Provincial Key Laboratory for Postharvest Physiology and Technology of Tropical Horticultural Products, Zhanjiang, Guangdong 524091, China

Received date: 2019-01-22

  Request revised date: 2019-05-08

  Online published: 2019-11-04

Copyright

Copyright reserved © 2019. Office of Acta Agronomica Sinica All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Medical Association or the Editorial Board, unless this is clearly specified.
Fold

Abstract

Caffeoyl-CoA-O methyltransferase (CCoAOMT) is a key enzyme in the lignin synthetic pathway and also plays an important role in abiotic stresses. According to the eggplant reference genome, SmCCoAOMT was cloned from eggplant. The maximum opening reading frame (ORF) of SmCCoAOMT was 564 bp, encoding 187 aa and its relative molecular weight was 20.95 ku. The bioinformation analysis indicated that SmCCoAOMT showed 90.67% sequence identity with CCoAOMTs of other plants and had all the conservative sequences in CCoAOMT. The expressions in eggplants with different peel colors and at different stages of fruit development were analyzed by qRT-PCR. The results showed that the expression of SmCCoAOMT was significantly higher in purplish red and blackish purple peels than in white and green peels, which was positively correlated with peel color. Comparing to control, on the 10 th and 20 th days after flowering (10DAF, 20DAF), the expressions of SmCCoAOMT were significantly down-regulated under high temperature (HT) stess, especially on 10DAF, the expression decreased to 9.23%. Which might lead to the light peel color. The results suggested that SmCCoAOMT positively regulated the peel coloring in eggplant under HT stress.

Key words： eggplant; SmCCoAOMT; high temperature stess; peel coloring

Cite this article

LYU Lingling , FENG Xuefeng , LI Ke , LI Wei . SmCCoAOMT Positively Regulates the Peel Coloring in Eggplant under High Temperature Stress[J]. Chinese Journal of Tropical Crops, 2019 , 40(10) : 2091 -2096 . DOI: 10.3969/j.issn.1000-2561.2019.10.025

茄子（Solanum melongena L.）,茄科茄属类蔬菜,起源于印度,中国为第2起源地[1]。茄子在我国南北均大面积栽培,但各地喜好的果形和颜色变化较大：在东北以绿色卵茄为主,华东地区主要是紫红线茄,在中原地区,主要以绿长茄为主,西南地区以紫茄为主,华南地区主要栽培紫红长茄[2]。因此,果皮颜色成为茄子重要的育种目标之一。茄子果皮着色的最佳温度为20~25 ℃,华南地区5—6月份的高温天气易造成茄子果皮颜色变浅甚至完全变白、着色不均匀等问题[3,4],严重影响了茄子的外观品质,同时降低了茄子的商品价值。
咖啡碱-辅酶A-O-甲基转移酶（caffeoyl-CoA- O methyltransferase, CCoAOMT）属于植物氧甲基转移酶I类,此类蛋白的氨基酸序列有8个保守的基序,其中基序A[(V/I/L)(V/L)(D/K)(V/I)GGXX(G/A)]、B[(V/I/F)(A/P/E)X(A/P/G)DAXXXK(W/ Y/F)]、C[(A/P/G/S)(L/I/V)(A/P/G/S)XX(A/P/G/S) (K/R)(V/I)(E/I)(L/I/V)]是植物氧甲基转移酶所共有,其余5个为CCoAOMT所特有[5],这类酶的相对分子量较小。CCoAOMT为木质素合成过程中的关键酶,参与植物的木质化进程。CCoAOMT基因在植物抵御生物、非生物胁迫过程中也发挥重要作用。西瓜在干旱胁迫下CCoAOMT蛋白表达上调[6]。玉米在干旱条件下CCoAOMT的活性被激活,表明木质素的生物合成与植物抗旱性强弱有关[7]。烟草[8]、水稻[9]、拟南芥[10]、龙眼[11]中CCoAOMT在调控植物对干旱、低温或高盐等逆境胁迫的生理适应反应中发挥重要作用。
本课题组前期研究中（文章未发表）,带花蕾的茄子植株经35 ℃高温（High temperature,HT）处理后进行转录组测序,发现高温下SmCCoAOMT在果实发育过程中显著下调表达,果皮颜色与常温对照（CK）相比也变浅,说明SmCCoAOMT与果皮着色密切相关。本文的目的是想阐明SmCCoAOMT在高温下茄子果皮着色中的作用,为全面了解高温下茄子果皮着色机理打下基础。

1 材料与方法

1.1 材料

以田间筛选出来的高温条件下果皮颜色变浅的紫红长茄30#为材料。在相同栽培管理条件下,选取大小一致的植株20株,在授粉当天,高温处理（35 ℃ 12 h/25 ℃ 12 h）下培养10株,对照（CK）（25 ℃ 12 h/20 ℃ 12 h）条件下10株,12 h/12 h光暗交替培养。分别于开花后10、15、20 d（day after flowering, 10DAF, 15DAF, 20DAF）采样,削取茄子果皮,每个样3次重复,共计18个样,液氮速冻后-80 ℃保存,作为转录组测序材料,同时用于基因表达分析。另外在田间种植4个不同果色的茄子品种：19#（白色果皮）、30#（紫红果皮）、49#（绿色果皮）、59#（紫黑果皮）,取成熟果实的果皮,3次重复,用于基因的表达分析。

1.2 方法

1.2.1 SmCCoAOMT的克隆 用北京天恩泽基因科技有限公司的柱式植物RNAout 2.0试剂盒（产品编号为CAT#: 90404-50）来提取茄子果皮的总RNA,对RNA进行纯度和浓度检测后,利用反转录试剂盒（TaKaRa）合成cDNA第1链,作为随后PCR扩增的模板。根据茄子基因组数据（http://eggplant.kazusa.or.jp/）,利用Primer premier 5.0设计SmCCoAOMT cDNA全长扩增引物,QC-F：5°-ATGACCACCTCTGCCGATGAAG-3°,QC-R：5°-TTAACTGATGCGGCGACAAAGA-3°。反应体系为：PCR缓冲液12.5 μL,8.4 μL灭菌去离子水,0.1 μL Taq酶,正向、反向引物各1 μL（10 μmol/L）,cDNA模板2 μL;PCR扩增条件为：94 ℃变性3 min;94 ℃变性0.5 min,58 ℃退火0.5 min,72 ℃延伸2 min,32个循环;72 ℃延伸10 min。将目的片段连接到pMD19-T上并转化大肠杆菌DH5α,阳性克隆送华大基因测序。
1.2.2 SmCCoAOMT的生物信息学分析 从NCBI上选取马铃薯、辣椒、番茄、烟草、可可树、茄子等植物的CCoAOMT氨基酸序列,利用DNAMAN软件分析其同源性。利用ClustalX 2与MEGA5软件和Neighbor-Joining方法构建茄子SmCCoAOMT与NCBI中已经登录的高等植物的CCoAOMT进化树。
1.2.3 SmCCoAOMT的表达分析 以18s rRNA为内参基因[12],对SmCCoAOMT基因在不同果皮颜色茄子品种及果实不同发育时期的表达进行qRT-PCR分析。定量取出1 µg RNA,使用TaKaRa定量反转录试剂盒进行反转录,得到的cDNA用作定量表达的模板。SmCCoAOMT定量表达的引物,dl-up：GGTGGGCTAATTGGCTATGA,dl-dn：AGGAAGCTGGCAGATTTCAA。反应体系：95 ℃ 30 s,1个循环;95 ℃ 5 s,55 ℃ 30 s,72 ℃ 30 s,40个循环。如果制作标准曲线,最后再添加一个溶解曲线的反应：95 ℃ 60 s,58 ℃ 30 s,95 ℃ 30 s,1个循环。

1.3 数据处理

采用SPSS 19.0软件对试验数据进行Duncan t检验。

2 结果与分析

2.1 SmCCoAOMT cDNA全长克隆及其生物信息学分析

通过PCR扩增和测序鉴定,得到SmCCoAOMT基因cDNA全长（图1）,其最大阅读框为564 bp,编码187个氨基酸（图2）,相对分子质量为20.95 ku。
图1 SmCCoAOMT基因PCR扩增

Fig. 1 PCR amplification of SmCCoAOMT

图2 茄子SmCCoAOMT基因的核酸序列及其氨基酸序列

Fig. 2 Nucleotide and putative amino acid sequences of the SmCCoAOMT

2.2 SmCCoAOMT的生物信息学分析

利用DNAMAN对马铃薯、辣椒、番茄、烟草、可可树、茄子等植物CCoAOMT氨基酸序列的同源性进行分析（图3）。结果发现茄子CCoAOMT与其他物种的CCoAOMT序列同源性高达90.67%,与马铃薯的同源性最高。同时茄子SmCCoAOMT具有CCoAOMT的全部保守序列（红色方框标示）。
图3 茄子SmCCoAOMT与其他植物CCoAOMT氨基酸序列同源性比对

红色方框内为植物CCoAOMT的保守序列元件。

Fig. 3 Alignment of SmCCoAOMT and other CCoAOMTs

Red-boxes are popular conserved sequence elements in plant CCoAOMTs.
NP001305508.1: Solanum tuberosum; AAF44689.1: Populus tomentosa; APX43200.1: Populus tomentosa; XP002298729.1: Populus trichocarpa; ALR35193.1: Fraxinus mandshurica; PON82039.1: Trema orientale; BAE48788.1: Codonopsis lanceolata; XP017233504.1: Daucus carota subsp. sativus; XP012831062.1: Erythranthe guttata; AAT75320.2:Boehmeria nivea; AAS91565.1: Broussonetia papyrifera; XP006856484.1: Amborella trichopoda; AFD23679.1: Salvia miltiorrhiza; XP007047308.1: Theobroma cacao; XP010263488.1: Nelumbo nucifera; XP010270267.1: Nelumbo nucifera; XP002282867.1: Vitis vinifera; XP016561061.1: Capsicum annuum var. grossum; ALP75647.1: Petunia × hybrid; XP019251878.1: Nicotiana attenuata; AFS33547.1: Nicotiana tabacum

利用ClustalX 2与MEGA5软件和Neighbor- Joining方法构建茄子SmCCoAOMT与NCBI中部分高等植物的CCoAOMT进化树,结果显示：茄子与甜椒、马铃薯最先归为一类,亲缘关系较近,其次是葡萄、烟草与矮牵牛,而与毛白杨的亲缘关系较远（图4）。
图4 SmCCoAOMT系统进化树分析

Fig. 4 Phylogenetic tree of SmCCoAOMT

NP001305508.1: Solanum tuberosum; AAF44689.1: Populus tomentosa; APX43200.1: Populus tomentosa; XP002298729.1: Populus trichocarpa; ALR35193.1: Fraxinus mandshurica; PON82039.1: Trema orientale; BAE48788.1: Codonopsis lanceolata; XP017233504.1: Daucus carota subsp. sativus; XP012831062.1: Erythranthe guttata; AAT75320.2: Boehmeria nivea; AAS91565.1: Broussonetia papyrifera; XP006856484.1: Amborella trichopoda; AFD23679.1: Salvia miltiorrhiza; XP007047308.1: Theobroma cacao; XP010263488.1: Nelumbo nucifera; XP010270267.1: Nelumbo nucifera; XP002282867.1: Vitis vinifera; XP016561061.1: Capsicum annuum var. grossum; ALP75647.1: Petunia × hybrid; XP019251878.1: Nicotiana attenuata ; AFS33547.1: Nicotiana tabacum.

2.3 SmCCoAOMT的表达分析

2.3.1 SmCCoAOMT在不同果色茄子品种中的表达 SmCCoAOMT在紫黑果皮59#品种中表达量最高,其次是紫红果皮30#品种,在绿色果皮49#和白色果皮19#中的表达量显著低于前2个品种（图5）,即在颜色深的茄子品种中的相对表达量高于在颜色浅的茄子品种中的表达量,说明SmCCoAOMT的表达量与茄子果色呈正相关。
图5 SmCCoAOMT在不同果色茄子中的表达

不同小写字母表示差异显著（P<0.05）。

Fig. 5 The expression of SmCCoAOMT in eggplants with different peel colors

Different lowercase letters indicate a significant difference determined by the Duncan test (P<0.05).

2.3.2 SmCCoAOMT在果实不同发育时期的表达 常温下,SmCCoAOMT在果实不同发育时期的表达量呈先降后升的趋势;高温下SmCCoAOMT的表达量在果实发育的3个时期变化不大,但与常温对照相比,在10DAF和20DAF时,其表达量都显著下降,尤其是在10DAF时,表达量仅为常温对照的9.23%。在果实发育早期,SmCCoAOMT表达量的显著下调可能是导致果皮着色不良的原因之一。

3 讨论

在茄科蔬菜苯丙烷类代谢过程中,含苯丙烷骨架的物质,如绿原酸、木质素、花青素、原花青素等都是以苯丙氨酸为合成前体[13]。此代谢途径有2大分支,都是以肉桂酸、香豆酸为中间产物,一类是在查尔酮合成酶（Chalcone synthase, CHS）的作用下进入花青素合成途径,另一类则是在CCoAOMT的作用下进入木质素合成途径（图7）,CHS和CCoAOMT就成了进入这2个途径的关键酶。
图6 SmCCoAOMT在果实不同发育时期的表达

不同小写字母表示差异显著（P<0.05）。

Fig. 6 The expression of SmCCoAOMT at different stages of fruit development

Different lowercase letters indicate a significant difference (P<0.05).

图7 花青素和木质素生物合成途径[14]

Fig. 7 Biosynthetic pathways of anthocyanin and lignin[14]

CHI: Chalcone isomerase; CHS: Chalcone synthase; PAL: Phenylalanine ammonialyase; DFR: Dihydroflavonol reductase; F3H: Flavanone 3-hydroxylase; ANS: Anthocyanidin synthase.

CCoAOMT所介导的甲基化反应使花青素酚羟基的反应活性降低、稳定性增强,使花青素不断积累,使花、果实等组织呈现各种不同的颜色[15]。李晓峰等[16]的研究发现,CCoAOMT的上调表达会使‘砀山酥梨’果皮由黄绿色变成褐色;psCCoAOMT是引起㮈果实褐变的主要基因之一[17];将PhCCoAOMT1转入只开白花的‘Mitchell’矮牵牛品种中,发现转基因植株叶片变紫,花色由白色变成粉红色[18]。本次研究中,SmCCoAOMT在颜色深的茄子品种中的相对表达量显著高于在颜色浅的茄子品种中的表达量;在高温胁迫下的果实发育过程中,SmCCoAOMT表达量下降,茄子果皮颜色也变浅,推测SmCCoAOMT是影响茄子果色形成的关键基因之一。Sugar等[19]的研究认为,在梨果实发育过程中,一旦受到逆境胁迫,CCoAOMT表达量就会下降,导致果实表皮细胞老化,果肉细胞增大不一致,使气孔保卫细胞破裂后形成的空洞内产生次生保护组织,导致细胞壁增厚,外层细胞破损,进而形成果点,造成果皮木栓化,从而引起果皮颜色变浅、有斑点、着色不均匀。本研究结果与其结果一致,但其具体的调控机制有待进一步研究。

References

Publishing order | Descend order by publishing year | Descend order by cited within
[1]
Kalloo G, Bergh B O. Genetic improvement of vegetable crops[M]. Oxford , UK: Pergamon Press, 1993.

[2]
吕玲玲, 李威, 肖熙鸥 . 茄子种质资源主要性状评价及其相关性分析[J]. 中国农学通报, 2016,32(4):165-170.

[3]
张志忠, 黄碧琦 . 23个茄子品种抗热性鉴定及其抗热机理的初步研究[J]. 热带作物学报, 2011,32(1):61-65.

[4]
易金鑫, 侯喜林 . 茄子耐热性遗传表现[J]. 园艺学报, 2002,29(6):529-532.

[5]
Joshi C P, Chiang V L . Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases[J]. Plant Molecular Biology, 1998,37(4):663-674.

[6]
Yoshimura K, Masuda A, Kuwano M , et al. Programmed proteome response for drought avoidance / tolerance in the root of a C3 xerophyte (wild watermelon) under water deficits[J]. Plant and Cell Physiology, 2008,49(2):226-241.

[7]
Riccardi A. Interpreting strategies and creativity// Beylard-Ozeroff A, Králová J, Moser-Mercer B. Translators’ stratigies and creativity. Amsterdam: John Benjamins Publishing Company, 1998: 171-179.

[8]
Maury S, Geoffroy P, Legrand M . Tobacco O-methyltransferases involved in phenylpropanoid metabolism. The different caffeoyl-coenzyme A/5-hydroxyferuloyl -coenzyme A 3/5-O-methyltransferase and caffeic acid/5-hydroxyferulic acid 3/5-O-methyl-transferase classes have distinct substrate specificifies and expression patterns[J]. Plant Physiology, 1999,121:215-224.

[9]
Zhao H Y, Sheng Q X, Lu S Y , et al. Characterization of three rice CCoAOMT genes[J]. Chinese Science Bulletin, 2004,49(15):1602-1606.

[10]
Kim J K, Bamba T, Harada K , et al. Time-course metabolic profiling in Arabidopsis thaliana cell cultures after salt stress trealment[J]. Journal of Experimental Botany, 2007,58(3):415-424.

[11]
陈虎, 何新华, 罗聪 , 等. 龙眼咖啡酰辅酶A-O-甲基转移酶(DlCCoAOMT)基因的克隆和表达分析[J]. 中国农业科学, 2012,45(1):118-126.

DOI

[12]
Gantasala N P, Papolu P K, Thakur P K , et al. Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L.)[J]. BMC Research Notes, 2013,6:312.

[13]
王星, 罗双霞, 于萍 , 等. 茄科蔬菜苯丙烷类代谢及相关酶基因研究进展[J]. 园艺学报, 2017,44(9):1738-1748.

[14]
Mellway R D, Tran L T, Prouse M B , et al. The wound-, pathogen-, and ultraviolet B-responsive MYB134 gene encodes an R2R3 MYB transcription factor that regulates proanthocyanidin synjournal in poplar[J]. Plant Physiology, 2009,150(2):924-941.

[15]
Winkel-Shirley B . Flavonoid biosynjournal. A colorful model for genetics, biochelIlistry, cell biology and biotechlology[J]. Plant Physiology, 2001,126(2):485-493.

[16]
李晓峰, 李雪, 贾兵 , 等. ‘砀山酥梨’褐皮芽变木质素含量及相关酶活性与CCoAOMT表达量分析[J]. 园艺学报, 2012,39(5):828-836.

[17]
陈桂信, 王玉珍, 赵利 , 等. 㮈果实CCoAOMT与LAR基因的筛选与表达分析[J]. 北方园艺, 2015(9):87-94.

[18]
Nur Fariza M S, Joëlle K M, Benjamin D W , et al . CCoAOMT Down-regulation activates anthocyanin biosynjournal in petunia[J]. Plant Physiology, 2016,170(2):717-731.

[19]
Sugar D, Basile S R . Color and russet variation among selections of ‘Bosc’ pear[J]. Journal of the American Pomological Society, 2008,62(2):77-81.

Options
Outlines

/

〈 〉

琼ICP备19001573号
Copyright © Chinese Journal of Tropical Crops, All Rights Reserved.
Tel: 0898-66989102　E-mail: rdzx136@163.com
Powered by Beijing Magtech Co. Ltd