桃4CL基因家族鉴定及其在果实色泽发育和采后贮藏冷害中的表达分析

doi:10.3969/j.issn.1004-1524.20221604

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (11): 2600-2610.DOI: 10.3969/j.issn.1004-1524.20221604

桃4CL基因家族鉴定及其在果实色泽发育和采后贮藏冷害中的表达分析

孔凡旺¹^,²(), 张志刚², 李伟², 陈玉峰², 王长江², 郑亚琴¹, 徐蒙¹^,^*()

1.临沂大学农林科学学院, 山东临沂 276000
2.邹城市自然资源和规划局, 山东济宁 273500

收稿日期:2022-11-11 出版日期:2023-11-25 发布日期:2023-12-04
作者简介:孔凡旺(1977—),男,山东邹城人,学士,高级工程师,主要从事林果病理和采后生理研究。E-mail: Zclcsdkfw@163.com
通讯作者: * 徐蒙,E-mail: xumeng@lyu.edu.cn
基金资助:
国家自然科学基金(32302621);山东省自然科学基金(ZR2021QC100);山东省现代农业产业技术体系(SDAIT-06-08);山东省高等学校青创科技计划创新团队(2021KJ055)

Identification of 4CL gene family in peach and its expression analysis in fruit coloration during development stage and chilling injury during post-harvest low temperature storage

KONG Fanwang¹^,²(), ZHANG Zhigang², LI Wei², CHEN Yufeng², WANG Changjiang², ZHENG Yaqin¹, XU Meng¹^,^*()

1. College of Agriculture and Forestry Sciences, Linyi University, Linyi 276000, Shandong, China
2. Zoucheng Natural Resources and Planning Bureau, Jining 273500, Shandong, China

Received:2022-11-11 Online:2023-11-25 Published:2023-12-04

摘要/Abstract

摘要：

4-香豆酸辅酶A连接酶(4-coumarate: CoA ligase, 4CL)在类黄酮和木质素等化合物代谢中具有重要作用。本研究基于桃基因组数据库系统鉴定4CL基因家族成员,从基因家族进化树的角度分析桃4CL基因家族的结构与潜在功能,结合转录组数据和qRT-PCR技术分析其在果实发育阶段类黄酮合成及采后冷害木质化调控中的功能。结果表明,基于桃基因组数据库鉴定出21个4CL基因家族成员,分布在8条染色体上;基因家族进化树分析表明,Pp4CL1与Pp4CL2属于第一亚家族,主要参与木质素合成,Pp4CL3-Pp4CL21属于第二亚家族,主要参与类黄酮的合成;启动子顺式元件分析发现其含有大量的非生物胁迫以及茉莉酸甲酯(MeJA)等激素响应元件。发育阶段的基因表达结果表明,Pp4CL8和 Pp4CL13的表达水平随发育阶段总体呈上升趋势,且在红肉品种中的表达量高于黄肉和白肉品种,猜测其可能参与花青素等类黄酮合成。采后低温贮藏阶段的基因表达结果表明,Pp4CL2可能在果实采后冷害木质化中发挥作用,程序性降温(low temperature conditioning,LTC)处理后Pp4CL8、Pp4CL10和Pp4CL14也可通过诱导类黄酮合成,减轻采后低温贮藏对果实造成的冷害。本研究将为桃果实色泽发育和采后保鲜提供理论依据。

关键词: 桃, 木质素, 黄酮, 基因表达

Abstract:

4CL (4-coumarate: CoA ligase) plays an important role in the metabolism of phenylalanine compounds such as flavonoids and lignin. In this study, the peach 4CL gene family members were systematically identified based on the peach genome database, and the structure and possible functions of the peach 4CLs were analyzed using the genetic family evolutionary analysis, combing transcriptome data and qRT-PCR techniques to analyze their functions in flavonoids synthesis at the fruit development stage and chilling-induced lignification regulation at postharvest. The results showed that 21 members of the 4CL gene family, namely Pp4CL1-Pp4CL21, were identified on 8 chromosomes based on the peach genome database. Phylogenetic analysis showed that Pp4CL1 and Pp4CL2 belong to the first subfamily, mainly involved in the synthesis of lignin, and Pp4CL3-Pp4CL21 belongs to the second subfamily, mainly involved in the synthesis of flavonoids. The cis-element analysis of the 4CL promoters found that it contains a large number of abiotic stress and MeJA-responsive and other hormone response elements. The gene expression of different peach varieties at the development stage showed that Pp4CL8 and Pp4CL13 increased generally, and higher expression was observed in blood-flesh varieties than in yellow-and milk-white-flesh varieties, suggesting that these genes may participate in the fruit flavonoids synthesis. The gene expression of peach fruit during post-harvest cold storage showed that Pp4CL2 may play a role in the chilling-induced lignification of fruit flesh after harvest. After low temperature conditioning (LTC) treatment, Pp4CL8, Pp4CL10 and Pp4CL14 may reduce the chilling injury of peach fruit during post-harvest cold storage by improving flavonoids content. This study provides a theoretical basis for the coloration of peach fruit and its postharvest preservation.

Key words: peach, lignin, flavonoids, gene expression

中图分类号:

S662.1

孔凡旺, 张志刚, 李伟, 陈玉峰, 王长江, 郑亚琴, 徐蒙. 桃4CL基因家族鉴定及其在果实色泽发育和采后贮藏冷害中的表达分析[J]. 浙江农业学报, 2023, 35(11): 2600-2610.

KONG Fanwang, ZHANG Zhigang, LI Wei, CHEN Yufeng, WANG Changjiang, ZHENG Yaqin, XU Meng. Identification of 4CL gene family in peach and its expression analysis in fruit coloration during development stage and chilling injury during post-harvest low temperature storage[J]. Acta Agriculturae Zhejiangensis, 2023, 35(11): 2600-2610.

图/表 11

表1 qRT-PCR基因表达引物序列

Table 1 Primer sequences for quantitative real-time PCR analysis

基因 Gene	正向引物 Forward primer (5'→3')	反向引物 Reverse primer (3'→5')
Pp4CL8 Pp4CL10 Pp4CL14 EF2	CCCACATACCCAATTGCCCA AAAGAGCGGCTACTGTCCAC TGGAATCTCTACATGCTCCTCAG AGCAGGCTCTTGGTGGTATCT	TGAGGGCAGGTTTGTTTGAGT GGAGGGAGAGAGCGAAATGG GGTGAGGTTAGCGTGAAGATGA GATTCAATGACGGGGAGGTAG

图1 桃4CL基因家族成员染色体分布情况桃4CL基因在染色体上的分布以红色字体突出显示,串联重复基因以灰色突出显示,片段复制以橘黄色直线连接。

Fig.1 Chromosome distribution of 4CL gene family members in peach The distribution of the peach 4CL gene on the chromosome is highlighted in red font, tandem repeat genes are highlighted in gray, and segment duplication is connected in orange straight lines.

表2 桃4CL基因家族成员蛋白理化性质

Table 2 Physical and chemical properties of 4CL gene family proteins in peach

基因名称 Gene name	登录号 GenBank	氨基酸数目 Amino acid	等电点 Isoelectric point	分子量 Molecular weight/u	不稳定系数 Instability index
Pp4CL1	XM_007222180.2	544	5.83	59 937.30	30.32
Pp4CL2	XM_007215457.2	551	5.84	59 981.39	37.07
Pp4CL3	XM_020570163.1	567	8.9	62 153.90	41.82
Pp4CL4	XM_020567696.1	572	6.32	62 409.91	38.71
Pp4CL5	XM_007219446.2	457	5.56	49 467.21	31.17
Pp4CL6	XM_007225695.2	548	6.06	59 512.92	36.62
Pp4CL7	XM_007203554.2	543	8.44	59 630.94	41.21
Pp4CL8	XM_007208395.2	542	8.63	59 168.68	37.81
Pp4CL9	XM_007222403.2	569	8.95	61 299.59	33.63
Pp4CL10	XM_007209025.2	557	7.26	61 211.61	53.40
Pp4CL11	XM_020569127.1	591	8.47	63 537.10	43.54
Pp4CL12	XM_007219420.2	569	5.73	61 733.01	42.35
Pp4CL13	XM_007209024.2	558	6.26	61 052.43	49.46
Pp4CL14	XM_020566011.1	635	6.56	69 746.76	36.41
Pp4CL15	XM_007221216.2	748	8.29	83 920.25	44.22
Pp4CL16	XM_020554905.1	748	8.29	83 920.25	44.22
Pp4CL17	XM_020554134.1	566	8.11	63 179.53	37.96
Pp4CL18	XM_020561814.1	555	7.23	60 667.33	38.26
Pp4CL19	XM_020564772.1	165	6.51	18 632.33	44.38
Pp4CL20	XM_007200387.2	218	6.6	25 006.86	38.89
Pp4CL21	XM_020570041.1	218	6.6	25 006.86	38.89

图2 桃4CL基因家族成员保守结构域分析与基因结构

Fig.2 Conservative domain analysis and gene structure of 4CL gene family members in peach

图3 桃4CL基因家族成员的保守基序

Fig.3 Conservative motifs of 4CL gene family members in peach

图4 桃4CL基因家族成员启动子的顺式作用元件

Fig.4 Cis-acting elements of peach 4CL gene family member promoters

图5 桃、杨树和拟南芥4CL基因家族成员共线性分析

Fig.5 Collinearity analysis of 4CL gene family members of peach, poplar and Arabidopsis

图6 桃4CL基因家族成员遗传进化分析

Fig.6 Phylogenetic analysis of 4CL gene family members in peach

图7 红肉、黄肉和白肉光核桃果肉发育阶段4CL基因表达模式 PH,硬核期;CE,细胞增大期;FR,果实成熟期。

Fig.7 4CL gene expression in the development stage of red, yellow and white flesh peaches PH,pit-harding; CE,cell enlargement; FR,fruit ripening.

图8 4CL基因家族成员在采后低温和LTC处理中的表达模式

Fig.8 Expression of 4CL gene family members in postharvest low temperature and LTC treatment

图9 qRT-PCR验证候选4CL基因在采后0 ℃(A)和LTC(B)处理中的相对表达水平

Fig.9 qRT-PCR verification of relative expression level of candidate 4CL genes at 0 ℃ (A) and LTC treatment (B)

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桃4CL基因家族鉴定及其在果实色泽发育和采后贮藏冷害中的表达分析

Identification of 4CL gene family in peach and its expression analysis in fruit coloration during development stage and chilling injury during post-harvest low temperature storage

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