板栗SWEET基因家族成员的鉴定及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0700

摘要/Abstract

摘要：

目的 SWEET基因家族与果实中淀粉含量、糖含量密切相关，分析板栗SWEET基因家族在板栗果实成熟过程中的表达模式，为后续研究板栗SWEET基因家族在果实成熟过程中的作用奠定基础。方法利用生物信息学方法鉴定出7种壳斗科物种SWEET基因家族，对其进行系统进化树构建、各个成员同源性分析、基因复制类型分析等；同时分析板栗SWEET基因家族蛋白理化性质、基因结构、选择压力、蛋白结构等；利用转录组和RT-qPCR分析板栗SWEET基因家族成员在不同果实成熟时期的表达模式。结果 7种壳斗科物种中共包含了129个SWEET基因家族成员，7种壳斗科物种分化之前共拥有16个SWEET基因家族成员。板栗SWEET基因家族共有19个，不均地分布在9条染色体和1条contig片段上；同时其蛋白拥有较为一致的Motif分布、保守结构域分布。顺势作用元件分析结果显示，大量的生长发育相关元件、激素响应元件以及胁迫响应元件存在于板栗SWEET基因家族启动子序列上。转录组和RT-qPCR分析显示，有9个板栗SWEET基因家族成员在板栗果实成熟过程中表达。其中CmSWEET1、CmSWEET3、CmSWEET4、CmSWEET16及CmSWEET19随着果实成熟过程基因表达量下调；CmSWEET2及CmSWEET9随着果实成熟过程基因表达量上调；CmSWEET15与CmSWEET17随着果实成熟过程基因表达量先下调再上调。结论共鉴定了129个壳斗科物种SWEET基因家族成员，其中有19个板栗SWEET基因家族成员，CmSWEET15与CmSWEET17表达模式与可溶性糖变化模式相似，其可能调控板栗果实成熟过程中可溶性糖转运；CmSWEET1、CmSWEET2、CmSWEET3、CmSWEET4、CmSWEET9、CmSWEET16及CmSWEET19表达模式与淀粉变化模式完全一致或完全相反，其可能调控板栗果实成熟过程中淀粉积累。

关键词: 板栗, 壳斗科, 基因家族, 淀粉, 可溶性糖, 表达模式, 生物信息学

Abstract:

Objective The SWEET gene family is closely related to the starch content and sugar content in the fruit. Focusing on the expression pattern of chestnut SWEET gene family during chestnut fruit ripening may lay the foundation for subsequent studies of the SWEET gene family function during fruit ripening in Chinese chestnut. Method Bioinformatics techniques was applied to identify the SWEET gene family of seven Fagaceae species. Phylogenetic tree was constructed on them, homology analysis of each member was performed, and gene duplication type analysis was performed, etc. The protein physicochemical properties, gene structure, selection pressure and protein structure of chestnut SWEET gene family were also analyzed. Transcriptome and RT-qPCR were used to analyze the expression patterns of chestnut SWEET gene family members during different fruit ripening period. Result A total of seven Fagaceae species contained the 129 members of SWEET gene family, and seven species had 16 SWEET gene family members before divergence. There were 19 chestnut SWEET gene families, which were unequally distributed across nine chromosomes and one contig segment. And their proteins had a relatively consistent Motif distribution and conserved domain distribution. The results of cis-element analysis showed that a large number of growth-related elements, hormone response elements and stress response elements were present on the promoter sequences of chestnut SWEET gene family. Transcriptome and RT-qPCR analyses revealed that nine members of the chestnut SWEET gene family were expressed during chestnut fruit ripening. Among them, CmSWEET1, CmSWEET3, CmSWEET4, CmSWEET16 and CmSWEET19 were downregulated with ripening, CmSWEET2 and CmSWEET9 were downregulated with ripening, and CmSWEET15 and CmSWEET17 were downregulated with ripening. Conclusion A total of 129 members of the Fagaceae species SWEET gene family are identified, including 19 members of the chestnut SWEET gene family.CmSWEET15 and CmSWEET17 expression pattern is similar to the change pattern of soluble sugars, which may regulate soluble sugar transport during chestnut fruit ripening. The expression patterns of CmSWEET1, CmSWEET2, CmSWEET3, CmSWEET4, CmSWEET9, CmSWEET16 and CmSWEET19 are completely consistent with or opposite to the pattern of starch changes, which may regulate starch accumulation during chestnut fruit ripening.

Key words: chestnut, Fagaceae, gene family, starch, soluble sugar, expression patterns, bioinformatics

杨涌, 袁国梅, 康肖肖, 刘亚明, 王东升, 张海娥. 板栗SWEET基因家族成员的鉴定及表达分析[J]. 生物技术通报, 2025, 41(2): 257-269.

YANG Yong, YUAN Guo-mei, KANG Xiao-xiao, LIU Ya-ming, WANG Dong-sheng, ZHANG Hai-e. Identification and Expression Analysis of Members of the SWEET Gene Family in Chinese Chestnut[J]. Biotechnology Bulletin, 2025, 41(2): 257-269.

图/表 14

表1 引物序列

Table 1 Primer sequences

基因Gene	上游引物Forward primer（5′-3′）	下游引物Reverse primer（5′-3′）
CmSWEET1	CTGCATGGTGTGGGCCTT	GATACCAGTGCCTGCCCC
CmSWEET2	GCGCTGTTTGTGTCACCC	AGCAGCTGAAGAAGGCGT
CmSWEET3	CAAAGGAAGCAGCCTCCCA	TGGGGCCAACTAGGGTGT
CmSWEET9	ATGCAGCTGGAGTCGCAG	CGGCAGCCCTGAGAACTG
CmSWEET14	GCTTTGCTGCTGCCGTTT	CAGGAAGTGCCGCACAGA
CmSWEET15	TCGCGTTTGTGCTCACCT	AAACGGCAGCAGCAAAGC
CmSWEET16	CAGCCGGGAGAGCTAGGA	AGCAGGGAGGTGCAAAGC
CmSWEET17	GCACCCGGGAGAGTTAGG	AGCAGGGAGGTGCAAAGC
CmSWEET19	CCTGGCAAAAGGCTCCCA	TGGCATGTACTCCACGCTC
CmActin	ATTCACGAGACCACCTACA	TGCCACAACCTTAATCTTCAT

图1 12个物种SWEET基因家族系统进化树图中不同分支颜色代表不同分组；基因名称的不同颜色代表不同同源性分组

Fig. 1 Phylogenetic tree of SWEET gene family of 12 speciesDifferent branch colors in the figure indicate different groups; different colors of gene names indicate different orthogroups

图2 各物种SWEET基因家族成员数量统计

Fig. 2 Statistics of the number of SWEET gene family members for each species

图3 各进化节点基因家族成员数量统计图中均为绿色方框代表各个节点SWEET基因家族成员数量；“+，-”后数字代表获得丢失的基因数量

Fig. 3 Statistics of the number of gene family members at each evolutionary nodeThe green boxes indicate the number of members of the SWEET gene family in each node. Numbers after “+, -” indicate the number of gained or lost genes

图4 CmSWEET基因家族的染色体定位

Fig. 4 Chromosomal localization of the CmSWEET gene family

图5 CmSWEET基因家族系统进化树（A）、motif分布（B）、结构域分布（C）和基因结构（D）

Fig. 5 Phylogenic tree (A), motif distribution (B), domain distribution (C), and gene structure (D) of CmSWEET gene family

图6 CmSWEET基因家族顺式作用元件统计

Fig. 6 Statistics of cis-acting elements of CmSWEET gene family

表2 七种壳斗科物种SWEET基因家族复制类型

Table 2 SWEET gene family duplication types in seven Fagaceae species

物种 Species	全基因组复制 WGD	串联复制 TD	近端复制 PD	分散复制 DSD	转座子复制 TRD	总数 Total
欧洲山毛榉 Fagus sylvatica	1	0	2	1	2	6
槲树 Quercus dentata	3	2	1	1	3	10
红锥 Castanopsis hystrix	2	4	3	0	5	14
钩椎 Castanopsis tibetana	1	1	2	0	4	8
日本栗 Castanea crenata	1	1	2	3	4	11
美洲栗 Castanea dentata	3	5	1	0	2	11
板栗 Castanea mollissima	0	5	1	3	3	12

图7 CmSWEET基因家族与基因组复制模式占比比较图中TRD、DSD、PD、TD及WGD代表转座子复制、分散复制、近端复制、串联复制及全基因组复制。下同

Fig. 7 Comparison of the proportion of CmSWEET gene family and genome replication typesTRD, DSD, PD, TD and WGD indicate transposon duplication, dispersed duplication, proximal duplication, tandem duplication and whole genome duplication in the figure. The same below

表3 基因对Ka/Ks值的计算

Table 3 Calculations of the gene-pairs Ka/Ks values

复制类型Duplication type	基因对Gene pair		非同义替换率Ka	同义替换率Ks	非同义替换率/同义替换率Ka/Ks
TD	CmSWEET5	CmSWEET6	0.44	4.81	0.09
TD	CmSWEET18	CmSWEET19	0.34	1.77	0.19
TD	CmSWEET6	CmSWEET7	0.37	1.07	0.34
TD	CmSWEET14	CmSWEET15	0.04	0.12	0.37
TD	CmSWEET4	CmSWEET5	0.22	0.45	0.49
PD	CmSWEET16	CmSWEET17	0.02	0.06	0.28
DSD	CmSWEET8	CmSWEET11	0.64	1.69	0.38
DSD	CmSWEET13	CmSWEET11	0.57	3.47	0.16
DSD	CmSWEET11	CmSWEET3	0.45	2.14	0.21
TRD	CmSWEET9	CmSWEET2	0.23	0.98	0.23
TRD	CmSWEET1	CmSWEET12	0.42	3.06	0.14
TRD	CmSWEET10	CmSWEET17	0.33	1.73	0.19

图8 CmSWEET基因家族不同复制模式蛋白结构比较

Fig. 8 Comparison of protein structures with different replication types of the CmSWEET gene family

表4 CmSWEET基因家族蛋白预测模型得分

Table 4 Prediction model scores of CmSWEET gene family proteins

蛋白编号Protein ID	预测模型得分pTM scores
CmSWEET1	0.84
CmSWEET2	0.86
CmSWEET3	0.62
CmSWEET4	0.75
CmSWEET5	0.81
CmSWEET6	0.77
CmSWEET7	0.75
CmSWEET8	0.69
CmSWEET9	0.87
CmSWEET10	0.70
CmSWEET11	0.81
CmSWEET12	0.87
CmSWEET13	0.85
CmSWEET14	0.79
CmSWEET15	0.79
CmSWEET16	0.73
CmSWEET17	0.72
CmSWEET18	0.70
CmSWEET19	0.72

图9 CmSWEET基因家族表达模式热图（A）、可溶性糖含量（B）和淀粉含量（C）变化趋势Nut70代表果实成熟初时期；Nut82代表果实成熟中期；Nut94代表果实成熟后期；不同小写字母表示在0.05水平差异显著；下同

Fig. 9 Heatmap of the CmSWEET gene family expression patterns （A）， the changing trend of soluble sugar content （B） and the starch content （C）Nut 70 indicates the early ripening period; Nut 82 indicates the middle ripening stage and Nut 94 indicates the late ripening stage. Different lower letters indicate significantly difference at the 0.05 level. The same below

图10 CmSWEET基因家族不同果实成熟时期相对表达量

Fig. 10 Relative expressions of the CmSWEET gene family during different fruit ripening periods

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