锦绣黄桃漆酶基因 <i>PpLAC7</i> 的克隆、亚细胞定位及表达分析

doi:10.7668/hbnxb.20194985

摘要/Abstract

摘要：

为克隆锦绣黄桃漆酶基因PpLAC7序列,分析PpLAC7基因序列信息及其在果肉褐变中的作用。以锦绣黄桃为试材,采用同源克隆方法从锦绣黄桃中获得漆酶基因PpLAC7的cDNA序列。采用生物信息学软件对PpLAC7基因的启动子元件、蛋白质理化性质、蛋白质二级、三级结构、系统发育进化树和同源氨基酸序列比对进行分析。此外还对PpLAC7基因进行亚细胞定位以及在果肉褐变过程中的表达规律进行了分析。生物信息学分析表明,PpLAC7的启动子区域含有光响应元件、茉莉酸响应元件、干旱响应元件、种子特异性调控等元件。PpLAC7基因全长为1 692 bp,其蛋白质共编码563个氨基酸,分子质量为61.867 ku,总原子数为8 621个,亲水系数为-0.028,理论等电点为5.95,不稳定指数为 36.48,脂溶性指数为 87.26。PpLAC7蛋白的α-螺旋、延长链、β-转角、无规则卷曲分别占氨基酸总数的 14.74%,28.77%,6.22%,50.27%。进化树结果显示,PpLAC7基因与苹果MdLAC7基因具有较高的同源性,PpLAC7基因的氨基酸序列同样含有3个典型的铜离子结构域。亚细胞定位结果表明,PpLAC7定位于内质网。PpLAC7基因在锦绣黄桃果肉褐变过程中呈大量上调表达。结合锦绣黄桃果肉褐变指数与PpLAC7表达量的关系,推测PpLAC7可能在锦绣黄桃果肉褐变过程中起重要的作用。

关键词: 锦绣黄桃, PpLAC7基因, 果肉褐变, 基因克隆, 生物信息学分析, 亚细胞定位

Abstract:

To clone the PpLAC7 gene sequence of Jinxiu yellow peach,investigating its gene sequence information and its role in flesh browning.The cDNA sequence of PpLAC7 was cloned from Jinxiu yellow peach by homologous cloning.The promoter region elements,physicochemical properties,secondary and tertiary structure,phylogenetic tree and amino acid sequence alignment of PpLAC7 gene were performed bioinformatics by using bioinformatics software.In addition,it also investigated the subcellular localization and the expression pattern of PpLAC7 during the process of fresh browning in Jinxiu yellow peach.The results of bioinformatics analysis indicated that the promoter region included light responsiveness,MeJA-responsiveness,drought-inducibility,seed-specific regulation elements,etc.The full length of PpLAC7 gene was 1 692 bp,encoding 563 amino acids,molecular weight was 61.867 ku,total number of atoms was 8 621,GRAVY was -0.028,theoretical pI was 5.95,instability index was 36.48,aliphatic index was 87.26.The secondary structure of PpLAC7 protein mainly contained α-helix of 14.74%,extended strand of 28.77%,β-turn of 6.22% and random coil of 50.27%.Phylogenetic analysis showed that PpLAC7 had high similarity to MdLAC7.Deduced amino acid sequence indicated that PpLAC7 protein,the same as other species,also contained three typical copper ion domains.Subcellular localization analysis revealed that PpLAC7 was located in endoplasmic reticulum.The expression of PpLAC7 gene was up-regulated during the storage of Jinxiu yellow peach fruit.Combining the relationship of flesh browning index and PpLAC7 expression,it indicated that PpLAC7 may play an important role in the browning process of Jinxiu yellow peach fruit.

Key words: Jinxiu yellow peach, PpLAC7 gene, Flesh browning, Gene cloning, Bioinformatics analysis, Subcellular localization

龚意辉, 张灿美, 周桂花, 陈玫羽, 谭倩, 皮水勤, 曾永贤. 锦绣黄桃漆酶基因 PpLAC7 的克隆、亚细胞定位及表达分析[J]. 华北农学报, 2024, 39(5): 72-79. doi: 10.7668/hbnxb.20194985.

GONG Yihui, ZHANG Canmei, ZHOU Guihua, CHEN Meiyu, TAN Qian, PI Shuiqin, ZENG Yongxian. Cloning,Subcellular Localization and Expression Analysis of PpLAC7 Gene from Jinxiu Yellow Peach[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(5): 72-79. doi: 10.7668/hbnxb.20194985.

http://www.hbnxb.net/CN/Y2024/V39/I5/72

图/表 10

表1 引物序列

Tab.1 The primer sequences

基因 Gene	上游引物(5'-3') Forward primer(5'-3')	下游引物(5'-3') Reverse primer(5'-3')	目的 Purpose
PpLAC7	ATGGCACGCTTTGCCTTCGT	CTAACATTGGGGTAGATCCG	基因克隆
PpTEF2	GGTGTGACGATGAAGAGTGATG	TGAAGGAGAGGGAAGGTGAAAG	内参引物
PpLAC7	GGCCCACAAGTTCAACACC	TTACGGGACACAGCTCCAGAT	实时荧光定量 PCR 引物

图1 锦绣黄桃PpLAC7基因cDNA的PCR 扩增 M.DL2000 DNA Marker;1.目标片段;2.对照。

Fig.1 Ampliation for the cloning of PpLAC7 genes cDNA in Jinxiu yellow peach M.DL2000 DNA Marker;1.The target fragment;2.The control.

表2 锦绣黄桃PpLAC7启动子元件预测

Tab.2 Structure of cis-elements in promoter region of PpLAC7 gene

序列 Motif	功能 Function	数量 Number
AAACCA	厌氧诱导的顺式作用调控元件	3
ATTAAT,GGTTAA,GGTTAAT,TGATAATGT,AACCTAA	光响应元件	10
CAAT,CCAAT,CAAAT	顺式作用元件	28
CGTCA,TGACG	茉莉酸响应元件	4
CAACTG	干旱响应元件	1
CATGCATG	种子特异性调控元件	1
TATA,ATATAT,TATATA,TATAAAA	核心启动子元件	28

图2 锦绣黄桃PpLAC7蛋白的二级结构分析蓝色、红色、绿色、橙色分别表示α 螺旋、延长链、β转角、无规则卷曲结构。

Fig.2 Secondary structure analysis of PpLAC7 protein in Jinxiu yellow peach Blue, red, green and orange represent alpha helix, extended strand, beta turn and random coil structure, respectively.

图3 锦绣黄桃PpLAC7蛋白三级结构预测分析

Fig.3 Tertiary structure predict of PpLAC7 protein in Jinxiu yellow peach

图4 锦绣黄桃PpLAC7蛋白系统发育树

Fig.4 Phylogenetic trees of PpLAC7 protein in Jinxiu yellow peach

图5 桃、苹果和荔枝漆酶氨基酸序列比对分析 Ⅰ、Ⅱ、Ⅲ分别为1个Ⅰ 型Cu^{2 +}、1个Ⅱ 型Cu^{2 +}、1个Ⅲ型Cu^{2 +} DNA结构域。

Fig.5 Amino acid sequence alignment analysis of laccase from peach,apple and litchi Ⅰ,Ⅱ and Ⅲ are the types of Ⅰ Cu²⁺, Ⅱ Cu²⁺, and Ⅲ Cu²⁺ of DNA domains, respectively.

图6 PpLAC7蛋白的亚细胞定位

Fig.6 Subcellular localization of PpLAC7

图7 PE包装对锦绣黄桃果肉褐变指数的影响不同大写字母表示不同处理间差异达极显著水平(P<0.01)。图8同。

Fig.7 Changes of browning index by PE treatment in Jinxiu yellow peach Different capital letters indicate extremely significant difference between different treatments(P<0.01).The same as Fig.8.

图8 PpLAC7在锦绣黄桃不同褐变程度果肉中的表达情况

Fig.8 Expression analysis of PpLAC7 during the process of flesh browning in Jinxiu yellow peach fruit

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锦绣黄桃漆酶基因 PpLAC7 的克隆、亚细胞定位及表达分析

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锦绣黄桃漆酶基因 PpLAC7 的克隆、亚细胞定位及表达分析

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