甜瓜CmEPF基因家族的鉴定及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0969

摘要/Abstract

摘要：

【目的】表皮模式因子（epidermal patterning factor, EPF）是一类植物特有的分泌蛋白，在植物生长发育特别是气孔形态建成过程中发挥着重要作用，旨为揭示甜瓜EPF基因的特性和功能。【方法】对甜瓜CmEPF基因家族进行基因组鉴定，利用生物信息学手段对其基因结构、系统进化以及组织表达特性进行分析。【结果】甜瓜基因组存在11个可分为3个亚家族的CmEPF成员，它们的基因结构相对保守，含有1-3个内含子，且大都分布于甜瓜的叶绿体上；基于系统进化分析将其分为3个亚家族。不同器官RT-qPCR分析发现，CmEPFs基因在甜瓜器官中广泛表达，但不同家族成员在不同器官中的表达模式存在差异，其在第二片叶表达量较高，且气孔密度、光合速率和蒸腾速率最高，其表达量与光合参数和气孔密度显著相关。【结论】CmEPFs基因通过影响甜瓜的气孔密度，影响其光合作用，从而调控甜瓜叶片气孔对外界环境的响应。

关键词: 甜瓜, 表皮模式因子, 拟南芥, 气孔, 叶绿体, 基因表达分析

Abstract:

【Objective】Epidermal patterning factor（EPF）is a unique secreted protein found in plants that plays a crucial role in plant growth and development, particularly in stomatal morphogenesis. The aims of this study was to gain insights into the characteristics and functions of the EPF gene in melon.【Method】We conducted a comprehensive analysis of the entire CmEPF family, including gene structure, phylogeny, and tissue expression, using bioinformatics tools. 【Result】Our findings revealed that the melon genome contained 11 members of the CmEPF family, which can be classified into three subfamilies. The gene structure of CmEPF was found to be relatively conserved, typically consisting of 1-3 introns, with most genes located in the melon chloroplast. Phylogenetic analysis further confirmed the division of CmEPF into three subfamilies. By performing RT-qPCR analysis on various organs, we observed widespread expression of the CmEPF gene in melon, albeit with different expression patterns among family members in different organs. Notably, the CmEPF gene had high expression in the second leaf, which was correlated with the highest stomatal density, photosynthetic rate, and transpiration rate. These results suggest that the expression level of CmEPF is closely associated with photosynthetic parameters and photosynthesis. 【Conclusion】Our findings provide evidence that the CmEPF gene can affect stomatal density in melon, thereby affecting photosynthesis and regulating stomatal response to the external environment in melon leaves.

Key words: melon, epidermal patterning factor, Arabidopsis, stomata, chloroplast, gene expression analysis

陈春林, 李白雪, 李金玲, 杜清洁, 李猛, 肖怀娟. 甜瓜CmEPF基因家族的鉴定及表达分析[J]. 生物技术通报, 2024, 40(4): 130-138.

CHEN Chun-lin, LI Bai-xue, LI Jin-ling, DU Qing-jie, LI Meng, XIAO Huai-juan. Identification and Expression Analysis of Epidermal Patterning Factor (EPF) Genes in Cucumis melo[J]. Biotechnology Bulletin, 2024, 40(4): 130-138.

图/表 10

表1 11个甜瓜CmEPFs基因的基本信息

Table 1 Basic information of 11 melon CmEPFs genes

基因Gene	序列号Sequence ID	蛋白长度Protein length/aa	分子量Molecular mass/kD	等电点pI	亚细胞定位Subcellular localization
CmEPF1	MELO3C019934	117	13 284.50	8.85	叶绿体Chloroplast
CmEPF2	MELO3C028633	95	10 118.80	8.48	叶绿体Chloroplast
CmEPFL1	MELO3C013075	115	12 993.08	8.95	叶绿体Chloroplast
CmEPFL2	MELO3C011120	153	17 572.13	9.44	细胞核Nucleus
CmEPFL3	MELO3C007746	134	14 956.48	8.88	质膜Plasma membrane
CmEPFL4	MELO3C007667	131	14 680.19	9.67	叶绿体Chloroplast
CmEPFL5	MELO3C021111	114	12 973.17	9.69	叶绿体Chloroplast
CmEPFL6	MELO3C014284	108	11 965.11	9.36	叶绿体Chloroplast
CmEPFL7	MELO3C017882	107	12 057.17	9.17	质膜Plasma membrane
CmEPFL8	MELO3C031683	152	17 238.58	6.58	叶绿体Chloroplast
CmEPFL9	MELO3C029250	122	13 519.80	9.13	叶绿体Chloroplast

图1 甜瓜和拟南芥EPF蛋白的系统进化分析红色、绿色和蓝色分别代表拟南芥、水稻和甜瓜的EPF蛋白

Fig. 1 Phylogenetic analysis of EPF protein in melon and Arabidopsis Red, green, and blue indicates EPF proteins from Arabidopsis, rice, and melon, respectively

图2 甜瓜CmEPFs基因结构分析

Fig. 2 Analysis of CmEPFs gene structure in melon

图3 甜瓜CmEPF蛋白的多重序列比对分析

Fig. 3 Multiplex sequence alignment analysis of melon CmEPF protein

图4 CmEPF蛋白保守结构域和保守基序分析

Fig. 4 Analysis in conserved domains and motifs of CmEPF protein

图5 甜瓜CmEPFs基因在不同器官中的表达模式

Fig. 5 Expression pattern of melon CmEPFs gene in different organs

图6 CmEPFs基因在甜瓜不同部位叶片中的表达分析 L0：甜瓜的心叶；L1：甜瓜从上到下第1片叶；L2：甜瓜从上到下第2片叶L3：甜瓜从上到下第3片叶L4：甜瓜从上到下第4片叶。不同小写字母表示处理间差异显著（P<0.05）。下同

Fig. 6 Expression analysis of CmEPFs gene in the leaves in different parts of melon L0: Heart leaf of melon; L1: melon from top to first leaf; L2: melon from top to second leaf; L3: melon from top to third leaf; L4: melon from top to fourth leaf. Different lowercase letters indicate significant difference among treatments（P<0.05）. The same below

表2 不同部位叶片的气孔形态

Table 2 Stomatal morphology of leaves in different parts

处理Treatment	气孔密度Stomatal density/（个·mm^-2）	气孔指数Stomatal index	气孔长度Stomatal length/μm	气孔宽度Stomatal width/μm
L0	649.8±87.9 a	0.240±0.028 a	17.59±0.41 d	13.48±0.33 a
L1	624.1±81.14a	0.257±0.024 a	20.26±0.52 c	13.26±0.57 a
L2	460.4±44.6 b	0.269±0.018 a	21.46±0.45b c	14.09±0.34 a
L3	351.0±19.2 b	0.282±0.011 a	22.47±0.44 ab	13.07±0.27 a
L4	391.9±38.7 b	0.285±0.018 a	23.06±0.79 a	13.54±0.33 a

图7 甜瓜不同部位叶片的光合气体参数

Fig. 7 Photosynthetic gas parameters of leaves in different parts of melon

图8 CmEPFs基因与光合参数、气孔密度、气孔指数的相关性分析 *表示处理间差异在P<0.05水平显著，**表示处理间差异在P<0.01水平显著

Fig. 8 Correlation analysis of CmEPFs gene with photosynthetic parameters, stomatal density and stomatal index * indicates that the difference between treatments is significant at P<0.05 level, ** indicates that the difference between treatments is significant at P<0.01 level

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