槜李APX基因家族的克隆与等位变异分析

doi:10.16178/j.issn.0528-9017.20240720

摘要/Abstract

摘要：

为鉴定分析槜李抗坏血酸过氧化物酶(ascorbate peroxidase, APX)基因家族,本研究以龙种槜李为研究材料,结合前期已取得的转录组数据,以中国李基因组序列为模板进行检索并设计引物,经过PCR、分子克隆、序列分析验证,成功分离出6个槜李APX基因(PsAPX-1a、PsAPX-1b、PsAPX-1c、PsAPX-1d、PsAPX-1e和PsAPX-1f)。NCBI检索发现,PsAPX-1f上存在1个氨基酸缺失位点,导致其不能形成完整的目标基因。将PsAPX-1a、PsAPX-1b、PsAPX-1c、PsAPX-1d、PsAPX-1e提交至GenBank,获得登录号分别为PP277487、PP277488、PP277489、PP277490、PP277491。多序列比对发现,PsAPX-1a、PsAPX-1b、PsAPX-1c、PsAPX-1d和PsAPX-1e的相似性为98.8%~99.7%。PsAPX-1a包含79 bp的3'UTR,3个外显子和2个内含子,不含5'UTR,存在1个1 656 bp的开放读码框,编码552个氨基酸。蛋白质结构预测表明,PsAPX-1a蛋白具有APX保守结构域,与青梅、杏树、桃树等物种具有较高的同源性,该蛋白序列与槜李的抗逆与开花有关。本研究结果对探究槜李抗坏血酸代谢调控分子机制提供了参考。

关键词: 槜李, APX基因, 基因克隆, 序列分析

Abstract:

In order to identify and analyze the gene family of ascorbate peroxidase in Prunus salicina, this study used Longzhong as the research material, combined with previously obtained transcriptome data, searched and designed primers using the Chinese plum genome sequence as a template. After PCR, molecular cloning, and sequence analysis verification, six APX genes in Prunus salicina were successfully isolated (PsAPX-1a, PsAPX-1b, PsAPX-1c, PsAPX-1d, PsAPX-1e and PsAPX-1f). An amino acid deletion site on PsAPX-1f was found by NCBI, which prevents PsAPX-1f forming a complete target gene. Submit PsAPX-1a, PsAPX-1b, PsAPX-1c, PsAPX-1d, and PsAPX-1e sequences to GenBank and obtain login numbers PP277487, PP277488, PP277489, PP277490, PP277491 respectively. Multiple sequence alignment revealed that the similarity between PsAPX-1a, PsAPX-1b, PsAPX-1c, PsAPX-1d, and PsAPX-1e ranged from 98.8% to 99.7%. It was found that PsAPX-1a contained a 3' UTR of 79 bp as well as 3 exons and 2 introns, 5' UTR was not included. There was an open reading frame of 1 656 bp encoding 552 amino acids. Protein structure prediction showed that the PsAPX-1a protein had an APX conserved domain, which had high homology with green plum, apricot, and peach. The protein sequence is related to the stress resistance and flowering of Prunus salicina. This study provides a reference for exploring the molecular mechanism of regulating ascorbic acid metabolism in Prunus salicina.

Key words: Prunus salicina, APX gene, gene cloning, sequence analysis

中图分类号:

S662.3

李彦彪, 汪骏翔, 袁梦婷, 陆其华, 刘洋. 槜李APX基因家族的克隆与等位变异分析[J]. 浙江农业科学, 2025, 66(12): 2956-2964.

LI Yanbiao, WANG Junxiang, YUAN Mengting, LU Qihua, LIU Yang. Cloning and allelic variation analysis of the APX gene family in Prunus salicina[J]. Journal of Zhejiang Agricultural Sciences, 2025, 66(12): 2956-2964.

图/表 15

表1 3 个基因克隆所用引物信息

Table 1 Primers used in cloning three genes

引物名称	引物序列(5'-3')	扩增区域/nt	PCR片段大小/bp
L1	L1-F:TTGACACAGATAGAGAGGTAACG	116~1 853	1 738
	L1-R:ACTCACTTACACGCGTACAG
L2	L2-F:TTGACACAGATAGAGAGGTAACG	116~1 852	1 737
	L2-R:CTCACTTACACGCGTACAGT
L3	L3-F:ATTGACACAGATAGAGAGGTAACG	116~1 853	1 738
	L3-R:ACTCACTTACACGCGTACAG

表2 差异候选基因注释信息和表达

Table 2 Annotation information and expression of differential candidate gene

基因编号	基因信息	上(下)调表达	差异表达倍数		特异表达组织	是否候选基因
基因编号	基因信息	上(下)调表达	双子房花	单子房花	特异表达组织	是否候选基因
gene1	serine/threonine-protein kinase HT1-like	上	3.0	1.0	叶片
gene2	hypothetical protein PRUPE_ppa015169mg	上	5.0	1.0	花瓣、子房、叶片、茎、果实
gene3	ultraviolet-B receptor UVR8	上	1.0	1.0	叶片
gene4	L-ascorbate oxidase homolog	下	0.3	1.0	花瓣、子房、叶片、茎、果实	是
gene5	general transcriptional corepressor trfA	上	1.5	1.0	叶片

图1 PsAPX-1基因总DNA电泳检测及PCR产物电泳检测 M表示DNA marker Ⅴ;1表示总DNA;L3表示PCR产物。

Fig.1 Electrophoresis of total DNA and PCR products of the PsAPX-1 gene

图2 PsAPX-1f氨基酸缺失位点示意图 “*”处代表缺失位点。

Fig.2 Schematic diagram of the amino acid deletion site on PsAPX-1f

表3 PsAPX-1基因DNA序列相似度

Table 3 DNA sequence similarity of PsAPX-1 gene 单位:%

基因	PsAPX-1a	PsAPX-1b	PsAPX-1c	PsAPX-1d	PsAPX-1e
PsAPX-1a		99.7	98.8	99.5	99.3
PsAPX-1b	0.3		98.9	99.6	99.4
PsAPX-1c	0.5	0.3		99.1	98.9
PsAPX-1d	0.5	0.4	0.2		99.6
PsAPX-1e	0.7	0.6	0.4	0.4

表4 PsAPX-1基因突变及插入/缺失位点

Table 4 Mutation and insertion/deletion sites of PsAPX-1 gene

基因	位点
基因	133	195	284	465	521	536	602	652	704	938	969	1 147	1 169	1 191	1 230	1 762	1 769	1 896	1 901	1 906	1 909
PsAPX-1a	A	T	A	G	T	G	G	T	A	T	A	T	—	—	—	A	A	A	G	G	T
PsAPX-1b	A	C	G	A	T	A	G	C	A	T	A	T	—	—	—	A	A	A	G	G	T
PsAPX-1c	A	C	G	A	T	G	G	C	A	T	A	T	C	C	G	G	A	G	A	A	G
PsAPX-1d	A	C	G	A	C	G	G	C	A	T	A	C	—	—	—	A	A	G	A	A	G
PsAPX-1e	G	C	G	A	T	G	A	C	G	A	G	T	—	—	—	A	G	G	A	A	G

图3 槜李PsAPX-1a基因结构黑色箭头代表外显子。

Fig.3 PsAPX-1a gene structure of Prunus salicina

图4 PsAPX-1a基因序列及氨基酸序列

Fig.4 Gene sequence and protein sequence of PsAPX-1a

图5 PsAPX-1a基因编码蛋白疏水性分析

Fig.5 Hydrophobicity analysis of PsAPX-1a gene encoded protein

图6 PsAPX-1a基因编码蛋白跨膜区预测

Fig.6 Prediction of transmembrane region of PsAPX-1a gene encoded protein

图7 PsAPX-1a基因编码蛋白与数据库目标蛋白(I1LN08.1.A)匹配结果

Fig.7 Matching results between PsAPX-1a gene encoded protein and database target protein (I1LN08.1.A)

图8 PsAPX-1a基因编码蛋白三级结构分析

Fig.8 Tertiary structure analysis of PsAPX-1a gene encoded protein

图9 PsAPX-1a基因编码蛋白保守结构分析 ascorbase即为抗坏血酸过氧化物酶基因。

Fig.9 Conserved structure analysis of PsAPX-1a gene encoded protein

图10 主要物种APX基因系统进化树

Fig.10 Phylogenetic tree of APX genes in major species

图11 主要物种APX蛋白系统进化树

Fig.11 Phylogenetic tree of APX proteins in major species

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