苹果ELO家族基因鉴定及其在低温胁迫下的表达分析

doi:10.16420/j.issn.0513-353x.2021-0545

摘要/Abstract

摘要：

为研究苹果ELO家族基因在蜡质合成及抗寒中的作用,利用生物信息的方法对苹果全基因组中的ELO家族成员进行鉴定与分析。结果表明,MdELO基因家族共包含7个成员,不均匀地分布在苹果的3条染色体（Chr2、Chr8和Chr15）上。MdELO蛋白包含211 ~ 305个不等的氨基酸残基,等电点在9.43 ~ 11.62之间。亚细胞定位结果表明,MdELO蛋白在细胞核、细胞膜和叶绿体中有分布。顺式作用元件分析表明,7个MdELO启动子上游2 000 bp序列分布有激素、环境适应性和逆境诱导等响应元件。进一步测定越冬期不同品种苹果枝条中的蜡质含量,结果显示,从初休眠期至萌芽期,蜡质含量呈逐渐下降趋势,且抗寒性强的‘俄矮元帅’蜡质含量显著高于‘宫崎短富’;从蜡质形态及枝条表皮形态来看,‘俄矮元帅’蜡质呈针芒状且分布紧密,其枝条表皮结构紧密,表面光滑,而‘宫崎短富’蜡质呈散块状,枝条表皮结构松散,表面粗糙;两种苹果枝条的脯氨酸含量、相对电导率和淀粉磷酸化酶活性随越冬期的延长呈先升后降的趋势,淀粉酶活性呈逐渐上升的趋势,其中‘俄矮元帅’各指标均高于‘宫崎短富’。综上表明‘俄矮元帅’抗寒性显著高于‘宫崎短富’。qRT-PCR分析发现,低温胁迫下苹果砧木垂丝海棠的叶和根中均能检测到ELO的表达,大部分基因表现出先升后降的趋势,在低温胁迫12或24 h表达量最高,说明该家族成员在植物抗寒中扮演着重要作用。

关键词: 苹果, 超长链脂肪酸, ELO基因家族, 生物信息分析, 表达分析, 抗寒性

Abstract:

In order to study the role of apple ELO gene family in wax synthesis and cold resistance,bioinformatics methods were used to identify and analyze the ELO family members in the whole apple genome. The results showed that the MdELO gene family consisted of seven members,which were unevenly distributed on three apple chromosomes（Chr2,Chr8 and Chr15）. The MdELO protein contains 211-305 amino acid residues with isoelectric points ranging from 9.43 to 11.62. The results of subcellular localization showed that MdELO protein was distributed in the nucleus,cell membrane and chloroplast. The analysis of cis-acting elements showed that the 2 000 bp sequences upstream of the seven MdELO promoters were distributed with response elements such as hormones,environmental adaptability and stress induction. The wax content in different apple cultivars was further determined,and the results showed that the wax content of‘Oregon Spur Delicious’was significantly higher than that of‘Miyazaki’;On the other hand,from the it perspective of wax morphology and cuticle morphology,the wax of‘Oregon Spur Delicious’was needle-like and closely distributed,and the cuticle structure of its branches was close and smooth,while the wax of‘Miyazaki’was loose and lumpy,and the cuticle structure of its branches was loose and the surface was rough. Additionally,the proline,relative electrical conductivity and starch phosphorylase of the two apple branches increased firstly and then decreased with the extension of the overwintering period,and the amylase increased gradually,among which the content and activity of all indexes of‘Oregon Spur Delicious’were higher than that of‘Miyazaki’. The changes of the above indexes showed that the cold resistance of‘Oregon Spur Delicious’was significantly higher than that of‘Miyazaki’. It was found that the expression of ELO family members can be detected in the roots and leaves through qRT-PCR analysis,and the expression level displayeded certain differences. Most of them showed a trend of rising first and then falling,and the expression level is the highest at 12 h or 24 h,indicating that this family members may play an important role in low temperature stress.

Key words: apple, ultra long chain fatty acid, ELO gene family, bioinformatics analysis, expression analysis, cold resistance

中图分类号:

S661.1

高彦龙, 吴玉霞, 张仲兴, 王双成, 张瑞, 张德, 王延秀. 苹果ELO家族基因鉴定及其在低温胁迫下的表达分析[J]. 园艺学报, 2022, 49(8): 1621-1636.

GAO Yanlong, WU Yuxia, ZHANG Zhongxing, WANG Shuangcheng, ZHANG Rui, ZHANG De, WANG Yanxiu. Bioinformatics Analysis of Apple ELO Gene Family and Its Expression Analysis Under Low Temperature Stress[J]. Acta Horticulturae Sinica, 2022, 49(8): 1621-1636.

图/表 12

表1 qRT-PCR引物序列

Table 1 qRT-PCR primer sequence

基因登录号 Accession No.	基因名称 Gene name	序列（5′-3′） Sequence
MD02G1155100	MdELO1	F：TCCTACGTGTACTACCTGTCCAGATTC	R：AGAGGAACGAGATGACGGTGAGG
MD08G1036600	MdELO2	F：CAGTCTACCTCATCTTCATGTCGTTCC	R：AGAGCAGAGTGGTGGAGAGTATGG
MD08G1151400	MdELO3	F：CCGCCATCGCCGTCTACATAAC	R：GGCTGGAATAGGTCCGAGAGGTAC
MD08G1227900	MdELO4	F：ATTTGGCTCCACACATCGCAGTC	R：CGGTCACCCTTCTCTTCCACTTTG
MD15G1269600	MdELO5	F：CCGCCATCGCCGTCTACATAAC	R：GGCTGGAATAGGTCCGAGAGGTAC
MD15G1421700	MdELO6	F：CAACCTCAACCTCCTCCTCCTCTC	R：GCAAACAGATAATCCAGAAGGGGTAGG
MD15G1421900	MdELO7	F：CACAGCCGTCCACAACCTCATC	R：AGGTGTTGGGTGGGAAGCAGAG

表2 苹果ELO基因家族理化性质分析

Table 2 Analysis of physicochemical properties of apple MdELO gene family

基因登录号 Accession No.	基因名称 Gene name	大小/aa Size	分子量 Molecular weight	等电点 Isoelectric point	亚细胞定位 Subcellular localization	不稳定系数 Instability index
MD02G1155100	MdELO1	305	34 129.36	10.18	质膜Plasma membrane	43.13
MD08G1036600	MdELO2	211	24 143.86	11.62	质膜/细胞核Plasma membrane/nucleus	75.39
MD08G1151400	MdELO3	302	33 869.14	10.23	质膜Plasma membrane	41.39
MD08G1227900	MdELO4	265	30 759.69	9.43	叶绿体Chloroplast	40.60
MD15G1269600	MdELO5	302	33 869.14	10.23	质膜Plasma membrane	41.39
MD15G1421700	MdELO6	270	31 346.38	9.50	叶绿体Chloroplast	38.86
MD15G1421900	MdELO7	273	31 159.93	9.53	叶绿体Chloroplast	38.90

图1 苹果ELO基因家族基因结构

Fig. 1 Gene structure analysis of ELO family in apple

图2 苹果ELO基因家族成员序列序列一致性为100%用深蓝色标记,> 75%用粉色标记,> 50%用浅蓝色标记。红色下划线代表ELO结构域。

Fig. 2 Multiple sequence alignment of apple MdELO gene family members Sequence identity of 100% is marked in dark blue,> 75% in pink,and > 50% in light blue. The red underline represents the ELO domain.

图3 苹果ELO基因家族进化及其三级结构 At：拟南芥;Sl：番茄;Cs：柑橘;Os：水稻。

Fig. 3 Phylogenetic tree and tertiary structure of apple MdELO gene family At：Arabidopsis thaliana;Sl：Solanum lycopersicum;Cs：Citrus reticulata Blanco;Os：Oryza sativa.

表3 MdELO基因的Ka/Ks

Table 3 Ka/Ks of MdELO gene

旁系同源基因对位点 Paralogous gene pair sites	Ka	Ks	Ka/Ks
MdELO1/MdELO2	0.427957	0.867576	0.493279
MdELO1/MdELO3	0.026568	0.236089	0.112535
MdELO1/MdELO4	0.755620	1.160104	0.651338
MdELO1/MdELO5	0.026568	0.236089	0.112535
MdELO1/MdELO6	0.745098	1.256016	0.593223
MdELO1/MdELO7	0.671194	1.008214	0.665726
MdELO2/MdELO3	0.368452	0.856895	0.429984
MdELO2/MdELO4	0.701287	1.012445	0.692667
MdELO2/MdELO5	0.368452	0.856895	0.429984
MdELO2/MdELO6	0.700478	1.012911	0.691550
MdELO2/MdELO7	0.743613	0.838943	0.886369
MdELO3/MdELO4	0.718204	1.073463	0.669053
MdELO3/MdELO5	0.701591	1.069425	0.656045
MdELO3/MdELO6	0.706619	1.203640	0.587069
MdELO3/MdELO7	0.636910	0.936570	0.680045
MdELO4/MdELO5	0.718204	1.073463	0.669053
MdELO4/MdELO6	0.018535	0.220872	0.083918
MdELO4/MdELO7	0.244042	0.942442	0.258947
MdELO5/MdELO6	0.706619	1.203640	0.587069
MdELO5/MdELO7	0.636910	0.936570	0.680045
MdELO6/MdELO7	0.246400	0.868520	0.283700

图4 苹果ELO基因家族的染色体定位

Fig. 4 Chromosomal localization of apple MdELO gene family

图5 苹果ELO基因家族启动子顺式作用元件分布

Fig. 5 Analysis of cis-acting elements in the promoters of apple MdELO gene family

图6 苹果MdELO基因之间（A）及苹果与拟南芥AtELO基因之间（B）的共线性

Fig. 6 Collinearity between apple MdELO genes（A）and between apple and Arabidopsis AtELO genes（B）

图7 ‘俄矮元帅’和‘宫崎短富’苹果越冬期蜡质含量（A）、蜡质形态（B）及枝条表皮形态（C）

Fig. 7 The wax content（A）,wax morphology（B）and branch epidermis morphology（C）of‘Oregon Spur Delicious’and‘Miyazaki’during wintering

图8 ‘俄矮元帅’和‘宫崎短富’不同越冬期枝条淀粉酶活性、淀粉磷酸化酶活性、脯氨酸含量和相对电导率的变化不同小写字母代表‘宫崎短富’苹果不同时期的差异显著性;不同大写字母代表‘俄矮元帅’苹果不同时期的差异显著性（P < 0.05）。

Fig. 8 Changes of amylase,starch phosphorylase,Proline and relative conductivity of‘Miyazaki’and ‘Oregon Spur Delicious’branches in different overwintering periods Different lowercase letters represent the significant differences of‘Miyazaki’at different times;Different capital letters represent the significant differences of‘Oregon Spur Delicious’at different times（P < 0.05）.

图9 低温胁迫下苹果砧木垂丝海棠根系（A）和叶片（B）中ELO基因的表达图中不同大写字母表示不同处理组之间的显著差异（P < 0.01）。

Fig. 9 Expression analysis of apple MhELO gene family in roots（A）and leaves（B）under low temperature stress Different uppercase letters represent significant differences between treatment groups under different root treatment times（P < 0.01）.

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