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作物学报 ›› 2022, Vol. 48 ›› Issue (11): 2826-2839.doi: 10.3724/SP.J.1006.2022.14172

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

鼓粒期遮光对黑绿豆种皮花青素积累及相关基因表达特性的影响

马超(), 冯雅岚, 吴姗薇, 张均, 郭彬彬, 熊瑛, 李春霞*(), 李友军*()   

  1. 河南科技大学农学院 / 河南省旱地农业工程技术研究中心, 河南洛阳 471000
  • 收稿日期:2021-09-24 接受日期:2022-03-25 出版日期:2022-11-12 网络出版日期:2022-04-20
  • 通讯作者: 李春霞,李友军
  • 作者简介:第一作者联系方式: E-mail: machao840508@163.com
  • 基金资助:
    本研究由国家自然科学基金项目(31401323);河南省自然科学基金项目(222300420430);河南省高等学校青年骨干教师培养计划项目(2021GGJS050)

Effects of shading at grain filling stages on anthocyanin accumulation and related gene expression characteristics in seed coat of black mung bean

MA Chao(), FENG Ya-Lan, WU Shan-Wei, ZHANG Jun, GUO Bin-Bin, XIONG Ying, LI Chun-Xia*(), LI You-Jun*()   

  1. Agronomy College, Henan University of Science and Technology / Dry-land Agricultural Engineering Technology Research Center in Henan, Luoyang 471000, Henan, China
  • Received:2021-09-24 Accepted:2022-03-25 Published:2022-11-12 Published online:2022-04-20
  • Contact: LI Chun-Xia,LI You-Jun
  • Supported by:
    The National Natural Science Foundation of China(31401323);The Natural Science Foundation of Henan Province(222300420430);The Training Program for University Young Key Teachers in Henan Province(2021GGJS050)

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摘要：

为明确黑绿豆种皮各花青素组分积累规律和遮光对其的影响, 以普通绿豆和黑绿豆为试验材料, 在大田栽培条件下于花后5 d开始对豆荚进行遮光处理。采用分光光度法结合液相色谱串联质谱的方法测定了2个绿豆种皮中不同时期各种花青素组分的积累, 利用Real-time PCR方法分析了花青素合成结构基因的表达, 并测定了3种花青素合成酶的活性。结果显示, 黑绿豆种皮在花后16 d (S3时期)开始大量积累花青素, 近成熟期时(S4时期)能检测到的花青素组分分别为: 飞燕草色素(88.67%, 主调色素)、矢车菊色素(8.68%)、原花青素(1.05%)、矮牵牛花色素(0.94%)、天竺葵色素(0.34%)和芍药花色素(0.32%)。其中, 3种主要花青素的主调色素组分分别为飞燕草素-3-O-葡萄糖苷(76.61%)、矢车菊素-3-O-葡萄糖苷(89.17%)、原花青素B3 (28.64%)。Real-time PCR分析表明, 黑绿豆种皮花青素合成结构基因PAL、C4H、4CL、CHS、CHI、F3’H、F3’5’H、DFR、LDOX和UFGT相对表达量显著高于普通绿豆, 其中DFR (371.85倍)、LDOX (44.09倍)和F3’5’H (32.99倍) 的差异表达倍数最大。黑绿豆种皮中PAL、CHI和UFGT的活性也均大于普通绿豆。9个花青素合成的结构基因表达量和2个花青素合成相关酶的活性与多数花青素组分含量和总含量都呈显著正相关关系。11个花青素合成结构基因的表达量和3个花青素合成相关酶活性在遮光条件下均显著降低, 最终导致花青素各组分含量和总量显著降低。研究结果为黑绿豆种皮着色的调控机制解析奠定了基础, 也有助于黑绿豆育种的辅助选择。

关键词: 黑绿豆, 遮光, 花青素, 种皮, 基因表达

Abstract:

To investigate the accumulation of anthocyanin in the seed coat of mung bean and the effects of shading on it, two cultivars with different seed colors (green and black) were both shaded after flowering 5 days under the conditions of field cultivation. The accumulation of various anthocyanin components in seed coat were assessed using spectrophotometry combined with liquid chromatography tandem mass spectrometer. The relative expressions of structural genes and the activity of three anthocyanin synthase enzymes were both analyzed. The results showed that the seed coat of black mung bean began to accumulate anthocyanins at 16 days after flowering (S3 period), and the detectable anthocyanins at the near-mature period (S4 period) were delphinidin (88.67%, main pigment), cyanidin (8.68%), procyanidin (1.05%), petunidin (0.94%), pelargonidin (0.34%), and peonidin (0.32%) according to the proportion of each component. In the black mung bean seed coat at S4 period, the main pigment components of the three anthocyanins were delphinidin-3-O-glucoside (76.61%), cyanidin-3-O-glucoside (89.17%), and procyanidin B3 (28.64%). In general, the relative expression levels of the anthocyanin synthesis structural genes (PAL, C4H, 4CL, CHS, CHI, F3’H, F3’5’H, DFR, LDOX, and UFGT) in the seed coat of black mung bean were significantly higher than green mung bean, among which DFR (37185%), LDOX (4409%), and F3’5’H (3299%) had the largest differential expression multiples. The activities of PAL, CHI, and UFGT in black mung bean seed coat were also greater than those of green mung bean. The relative expression levels of nine anthocyanin synthesis structural genes and the activity of two anthocyanin synthesis-related enzymes were dramatically positively correlated with the contents and the total contents of most anthocyanin components. The relative expression levels of eleven anthocyanin synthesis structural genes and the activities of three anthocyanin synthesis-related enzymes were remarkably reduced under shading conditions, which ultimately led to a noticeable decrease in the content and total amount of anthocyanin components. The results provide a theoretical foundation for the regulation mechanism of black mung bean seed coat coloration, and also contribute to the auxiliary selection of black mung bean breeding.

Key words: black mung bean, shading, anthocyanin, seed coat, the relative expression levels of genes

图1

普通绿豆和黑绿豆成熟种子表型"

表1

引物序列"

基因名称
Gene name		 正向引物
Forward primer (5′-3′)		 反向引物
Reverse primer (5′-3′)
VrPAL TCTTCATCCTATCACCCCACA TCATGCTATCAATCACCCAATC
VrC4H ATACTAAAGCATTCCACCGTT AGATACATCACCACCTACCCA
Vr4CL GCTGGAAGAAGAACGCTTGTT GTTGTTGGATTGAATGGTGGG
VrCHS GTCCATTCCCAAACCACCTCA TCGGCACTTCCACAACCACTA
VrCHI AAGCTAATAAGAGGATCGAAG AGAGAAACTAAGCCCCAAG
VrF3H CAAATCAAAGGAACCACAAG TTATCACAATCATACACAACAACT
VrF3'H GGTGGTGGAGGTGATGGTG ACTTGGGTCAGGATTCTTGGA
VrF3'5'H CAAAGTGATAGGTAGGGAGCG GGTTGAGAGGAGTTGAAGGGT
VrDFR AATGAAGTGATAAAGCCGACA GCATAAGAAAGGGACCAACG
VrLDOX AGGAGTTGGAGAACATAGGAA AGGATAAACAAGGTGGAAGAA
VrUFGT GGTGTGAGGATAAGATGGTTGT TGCGAGGTAGGGAATGAAGT
VrACTIN GTTCTGTTCCAGCCATCCAT GTGGTGCGACAACCTTGATT

图2

遮光条件下普通绿豆和黑绿豆种皮不同时期的表型分析 S1: 花后10 d; S2: 花后13 d; S3: 花后16 d; S4: 花后19 d。"

图3

遮光对普通绿豆和黑绿豆种皮花青素含量的影响 A: HCl-甲醇法花青素含量; B: LC-MC/MC法花青素含量; C: HCl-甲醇法和LC-MC/MC法花青素含量结果的相关性分析。数值为3个生物学重复的平均值, 误差线为标准误。同一个处理下不同小写字母代表不同时间差异显著。GS1: 普通绿豆花后10 d; GS2: 普通绿豆花后13 d; GS3: 普通绿豆花后16 d; GS4: 普通绿豆花后19 d; BS1: 黑绿豆花后10 d; BS2: 黑绿豆花后13 d; BS3: 黑绿豆花后16 d; BS4: 黑绿豆花后19 d。"

图4

遮光对普通绿豆和黑绿豆种皮花青素组分比例的影响 A: 光照条件下; B: 遮光条件下。缩写同图3。"

表2

遮光对普通绿豆和黑绿豆种皮飞燕草色素组分相对含量的影响"

表3

遮光对普通绿豆和黑绿豆种皮矢车菊色素组分相对含量的影响"

表4

遮光对普通绿豆和黑绿豆种皮原花青素组分相对含量的影响"

图5

遮光对普通绿豆和黑绿豆种皮中花青素结构基因表达特性的影响 缩写同图3。数值为3个生物学重复的平均值, 误差线为标准误。同一个处理下不同小写字母代表不同时间差异显著(P < 0.05)。"

图6

遮光对普通绿豆和黑绿豆种皮中花青素合成相关酶活性的影响 缩写同图3。数值为3个生物学重复的平均值, 误差线为标准误。同一个处理下不同小写字母代表不同时间在0.05水平差异显著。"

表5

花青素含量与相关基因表达及酶活性的相关系数"

指标
Index		 总花青素
Total
anthocyanin		 飞燕草色素
Delphinidin		 矢车菊色素
Cyanidin		 矮牵牛色素
Petunidin		 原花青素
Procyanidine		 天竺葵色素
Pelargonidin		 芍药花色素
Peonidin
基因相对表达量
Relative expression
level of genes		 PAL 0.97** 0.99** 0.98** 0.99** 0.33 0.63 0.82**
C4H 0.89** 0.96** 0.92** 0.95** 0.46 0.73* 0.90**
4CL 0.93** 0.98** 0.95** 0.98** 0.37 0.66 0.86**
CHS 0.74* 0.84** 0.78** 0.84** 0.61 0.80** 0.91**
CHI 0.37 0.50 0.42 0.50 0.85** 0.86** 0.83**
F3H 0.01 0.02 -0.01 0.02 0.73* 0.62 0.45
F3'H 0.99** 0.98** 0.99** 0.99** 0.33 0.63 0.80**
F3'5'H 0.92** 0.93** 0.91** 0.94** 0.43 0.69 0.86**
DFR 0.94** 0.94** 0.93** 0.97** 0.42 0.69 0.85**
LDOX 0.93** 0.94** 0.93** 0.96** 0.44 0.70* 0.86**
UFGT 0.84** 0.82** 0.83** 0.83** 0.49 0.72* 0.81**
酶活性
Activity
of enzymes		 PAL 0.87** 0.93** 0.88** 0.91** 0.42 0.69 0.87**
CHI 0.43 0.08 0.01 0.08 0.65 0.54 0.44
UFGT 0.77** 0.89** 0.83** 0.87** 0.44 0.69 0.87**
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