枸杞<i>LbaHY5</i>基因克隆、亚细胞定位及表达分析

doi:10.7668/hbnxb.20195209

摘要/Abstract

摘要：

为研究枸杞LbaHY5基因的性质及功能,探明LbaHY5在枸杞果实发育中的作用,以宁夏枸杞宁杞1号为试材,采用生物信息学、亚细胞定位及qRT-PCR等方法对LbaHY5的结构和功能进行初步分析,结果表明:LbaHY5开放阅读框全长为483 bp,编码160个氨基酸,分子质量为17.52 ku,等电点为9.69,属于亲水性蛋白。保守结构和多序列比对分析显示,LbaHY5蛋白含有bZIP结构域,属于bZIP家族成员。进化树分析表明,枸杞LbaHY5蛋白与茄科植物如番茄、马铃薯等的HY5蛋白高度同源。此外,启动子分析表明,该基因启动子区域富含多种功能元件,这些元件分别与光信号、激素信号以及非生物胁迫应答等过程相关。亚细胞定位结果显示,LbaHY5蛋白定位在细胞核上。qRT-PCR检测发现,LbaHY5基因在花中表达量最高,茎中最低,且在果实发育时期表达量呈现逐渐上升趋势。研究表明LbaHY5可能参与枸杞花器官和果实生长发育。

关键词: 枸杞, LbaHY5基因, 基因克隆, 亚细胞定位, 表达分析

Abstract:

To investigate the characterization and function of LbaHY5 gene of Lycium barbarum,explore the role of LbaHY5 in the degree of stem straightening in Lycium barbarum.Using Ningqi No.1 as the test material,a preliminary analysis was conducted on the structure and function of the LbaHY5 gene using bioinformatics,subcellular localization,and qRT-PCR methods,the results indicated that the LbaHY5 gene cloned from Ningqi No.1 had a full-length open reading frame of 483 bp,encoding 160 amino acids,with a molecular weight of 17.52 ku and an isoelectric point of 9.69,classifying as a hydrophilic protein.Conserved domain and multiple sequence alignment analysis showed that LbaHY5 protein contained a type bZIP domain,which belonged to the bZIP gene family.Evolutionary tree analysis showed that the LbaHY5 protein in goji berries was highly homologous to the HY5 protein in Solanaceae plants such as tomatoes and potatoes.In addition,promoter analysis showed that the promoter region of this gene was rich in multiple functional elements,which were respectively related to processes such as light signaling,hormone signaling pathways,and abiotic stress response.The subcellular localization indicated that LbaHY5 was located on the nucleus.The transcript level of LbaHY5 gene was the highest in the flower and the lowest in the stem.During the fruit development period,the expression level of LbaHY5 gradually increased.The results speculated that LbaHY5 gene might play an important role in the growth and development of floral organs and fruits in Lycium barbarum.

Key words: Lycium barbarum, LbaHY5 gene, Gene cloning, Subcellular localization, Expression analysis

赵安琪, 尹跃, 何军, 安巍, 秦小雅, 胡体旭. 枸杞LbaHY5基因克隆、亚细胞定位及表达分析[J]. 华北农学报, 2024, 39(6): 76-83. doi: 10.7668/hbnxb.20195209.

ZHAO Anqi, YIN Yue, HE Jun, AN Wei, QIN Xiaoya, HU Tixu. Cloning,Subcellular Localization and Expression Analysis of LbaHY5 Gene from Lycium barbarum[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(6): 76-83. doi: 10.7668/hbnxb.20195209.

http://www.hbnxb.net/CN/Y2024/V39/I6/76

图/表 12

图1 宁杞1号形态特征 A.整株;B.花;C.果实;D.果实发育时期。

Fig.1 Morphological characteristics of Ningqi No.1 A.Whole plant;B.Flower;C.Fruit;D.Fruit development period.

表1 本试验所需引物

Tab.1 Primers used in this study

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	用途 Purpose
LbaHY5-F	ATGCAAGAGCAAGCGACAAGTTC	基因克隆
LbaHY5-R	TTACTTCCTCCCTTCCTGAGCAC
GFP-LbaHY5-F	GAGAACACGGGGGAC TCTAGAATGCAAGAGCAAGCGACAAGTTC	亚细胞定位
GFP-LbaHY5-R	GTCCTTGTAGTCCAT GGTACCCTTCCTCCCTTCCTGAGCAC
qLbaHY5-F	GCGACAAGTTCCATTGCAGCTAG	定量引物
qLbaHY5-R	TCCACCCATCTCTGGCACTCTTC
Actin-F	CAATCGGGTATTTCAAGGTCAAG	内参基因
Actin-R	GAGCAGTGTTTCCCAGCATTG

图2 LbaHY5基因PCR扩增 M.DNA Marker;1,2.宁杞1号LbaHY5 CDS全长。

Fig.2 PCR amplification of LbaHY5 gene M.DNA Marker;1,2.The total length of LbaHY5 CDS of Ningqi No.1.

图3 LbaHY5的核苷酸序列及编码的氨基酸序列

Fig.3 Nucleotide sequence and encoded amino acid sequence of LbaHY5

图4 LbaHY5蛋白的保守结构域分析 A.保守结构域分析;B.多重序列比对;C.保守基序预测。

Fig.4 A putative conserved domain of LbaHY5 amino acid sequence A.Conservative structural domain analysis;B.Multiple sequence alignment;C.Conservative motif prediction.

图5 LbaHY5基因的染色体定位(A)和基因结构分析(B)

Fig.5 Chromosome localization(A)and gene structure analysis(B)of LbaHY5 gene

图6 LbaHY5蛋白二级结构预测分析蓝色、紫色和绿色分别代表α-螺旋、无规则卷曲和β-转角。

Fig.6 Prediction analysis of secondary structure of LbaHY5 protein Blue,purple,and green represent α-helix, random coil,and β-turn,respectively.

图7 LbaHY5蛋白的三级结构预测分析

Fig.7 Predicted three-dimension structure of LbaHY5 protein

图8 宁杞1号与其他物种HY5蛋白序列的系统进化树

Fig.8 The phylogenetic tree of HY5 protein from Ningqi No.1 and other plants

表2 LbaHY5基因启动子顺式作用元件分析

Tab.2 Cis-acting elements of LbaHY5 gene promoter

元件名称 Element name	核心序列 Core sequence	元件数目/个 Element number	功能 Function
G-box	TACGTG	1	参与光响应顺式作用元件
G-box	CACGTT	2	参与光响应顺式作用元件
TCT-motif	TCTTAC	4	部分光响应元件
AE-box	AGAAACAA	1	参与光响应顺式作用元件
O2-site	GTTGACGTGA	1	参与玉米醇溶蛋白代谢调节的顺式作用调节元件
ARE	AAACCA	1	厌氧诱导必需元件
ABRE	ACGTG	2	参与脱落酸反应的顺式作用元件
TGACG-motif	TGACG	4	参与茉莉酸甲酯反应的顺式作用调节元件
CGTCA-motif	CGTCA	4	参与茉莉酸甲酯反应的顺式作用调节元件
TATC-box	TATCCCA	1	参与赤霉素反应的顺式作用元件
TGA-box	TGACGTAA	1	参与生长素响应的顺式作用元件
MBS	CAACTG	1	参与干旱诱导的MYB结合位点

图9 LbaHY5蛋白亚细胞定位

Fig.9 Subcellular localization of LbaHY5 protein

图10 LbaHY5在枸杞不同组织(A) 及果实发育时期(B)的表达模式不同字母表示在P<0.05水平差异显著。

Fig.10 Expression analysis of LbaHY5 in different tissues(A)and fruit developmental stages(B)in goji Different letters indicate significant differences at the 0.05 level.

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枸杞LbaHY5基因克隆、亚细胞定位及表达分析

Cloning,Subcellular Localization and Expression Analysis of LbaHY5 Gene from Lycium barbarum

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