黄瓜<i>CsGDSL</i>脂解酶基因的克隆与表达分析

doi:10.7668/hbnxb.20194079

摘要/Abstract

摘要：

GDSL脂解酶是影响角质层发育的重要基因,克隆黄瓜GDSL脂解酶基因,并分析其表达模式,旨在为黄瓜角质层发育及黄瓜果实光泽的研究奠定基础。根据黄瓜基因组数据库中的参考序列,对黄瓜GDSL脂解酶基因进行克隆,并进行生物信息学分析。利用qRT-PCR技术明确该基因在黄瓜各组织部位中的表达情况。以6份光泽性不同的黄瓜为材料,对该基因序列进行克隆,以明确该基因在不同光泽性黄瓜中的作用。对黄瓜GDSL脂解酶基因启动子进行克隆并分析其作用元件。参照黄瓜基因组数据库设计扩增引物进行PCR扩增,获得CsGDSL基因,CDS长度1 059 bp,编码352个氨基酸,二级结构以无规卷曲(45.45%)与α-螺旋(33.24%)为主;该基因在进化过程中较为保守,与甜瓜CmGDSL亲缘关系最为紧密;在黄瓜花后3 d的子房中表达量较开花0 d的子房高;在6个品种中CsGDSL基因CDS序列保守; CsGDSL基因与逆境胁迫、激素及光等具有响应。获得了CsGDSL脂解酶基因,明确了其在黄瓜不同组织部位中的表达情况,该基因在不同材料中较为保守,由此推测,黄瓜CsGDSL可能影响黄瓜果皮光泽。

关键词: 黄瓜, 角质层, GDSL基因, 脂解酶

Abstract:

GDSL is an important gene affecting the development of cuticle.This study cloned cucumber GDSL lipase gene and analyzed its expression pattern,in order to lay a foundation for the study of cuticle development and the glassiness of cucumber.The gene sequence was cloned to clarify its role in cucumbers with different glossiness by using six cultivars with different glossiness as materials.The promoter of CsGDSL lipase gene was cloned to analyze its functional elements.According to the reference sequence in cucumber genome database,we cloned the GDSL lipase gene of cucumber and analyzed by bio-informatics.The expression of the gene in different tissue parts of cucumber was confirmed by qRT-PCR technique.The CDS of CsGDSL length 1 059 bp which encoded 352 amino acids,and the secondary structure was mainly random curl(45.45%)and α-helix(33.24%).This gene was conservative in the process of evolution and was most closely related to CmGDSL.The expression level was the highest in the male flower of cucumber on the flowering day,and the expression level in the ovary at 3 days after flowering was higher than that at 0 days after flowering.The CDS sequence of CsGDSL gene was conservative in six cultivars. CsGDSL gene was responsive to stress,hormone and light.We obtained the CsGDSL lipase gene in this study,and identified its expression in different tissue parts of cucumber,the gene is relatively conserved in different cultivars,suggesting that CsGDSL may affect cucumber glossiness.

Key words: Cucumber, Cuticle, GDSL gene, Lipase

高璐瑶, 曹嘉健, 王春华, 武涛, 杜亚琳. 黄瓜CsGDSL脂解酶基因的克隆与表达分析[J]. 华北农学报, 2023, 38(4): 47-53. doi: 10.7668/hbnxb.20194079.

GAO Luyao, CAO Jiajian, WANG Chunhua, WU Tao, DU Yalin. Cloning and Expression Analysis of CsGDSL Lipase Gene in Cucumber[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(4): 47-53. doi: 10.7668/hbnxb.20194079.

http://www.hbnxb.net/CN/Y2023/V38/I4/47

图/表 8

表1 引物信息

Tab.1 Primer information

引物名称 Primer code	引物序列(5'—3') Primer sequence (5'—3')	用途 Purpose
GDSL-F	ATGGAGTCAACAAAGCTCTTCCT	基因CDS扩增
GDSL-R	TTAGAAATGCTGTTTGAAATTGGA
GDSL-pro-F	TAAAGATTGAAAGACGACATGTGC	基因启动子扩增
GDSL-pro-R	ACAAGTTTGGTTCTGTATCTCCCA
qGDSL-F	CACCATCACGTCCATAGCCA	qRT-PCR
qGDSL-R	CGAAATCGCTCAAAACGCGA
Csactin-F	CGCTCTTCTTGCTTTCAC	qRT-PCR
Csactin-R	TACCTTGCCTTGGAG

图1 黄瓜CsGDSL基因CDS的克隆 M.DM2000 DNA Marker;1.目的片段。

Fig.1 Cloning the CDS of CsGDSL gene in cucumber M.DM2000 DNA Marker;1.Target fragment.

图2 黄瓜CsGDSL蛋白序列分析 A.CsGDSL二级结构预测: .螺旋, .折叠,➡.卷曲;B.CsGDSL三级结构预测。

Fig.2 Sequence analysis of CsGDSL protein in cucumber A.CsGDSL secondary structure prediction: .Helix, .Strand,➡.Coil;B.Prediction of the tertiary structure of CsGDSL.

图3 CsGDSL生物信息学分析 A.CsGDSL氨基酸序列同源比对;红框中为保守结构域;B.CsGDSL进化发育树分析。

Fig.3 Bio-informatics analysis of CsGDSL A.CsGDSL amino acid sequence homologous alignment;The red box represents the conserved domain;B.CsGDSL phylogenetic tree analysis.

图4 CsGDSL在黄瓜各组织部位中表达模式分析不同小写字母表示处理时间之间在0.05水平存在显著差异。

Fig.4 The expression pattern of CsGDSL in different tissue parts in cucumber Different lowercase letters indicate that significant difference between the treatment times at the level of 0.05.

图5 6个品种中CsGDSL核苷酸序列分析

Fig.5 Analysis the nucleotide sequence of CsGDSL in six cultivars

图6 黄瓜CsGDSL基因启动子的克隆 M.DM 2000 DNA Marker;1.目的片段。

Fig.6 Cloning the promoter of CsGDSL gene in cucumber M.DM 2000 DNA Marker;1.Target fragment.

表2 CsGDSL启动子调控元件分析

Tab.2 Analysis of regulatory elements in the promoter of CsGDSL

位点名称 Name	CsGDSL	序列 Sequence	功能 Function
A-box	-	CCGTCC	顺式调节元件
AAGAA-motif	+	gGTAAAGAAA
ABRE	-	ACGTG	顺式作用元件参与脱落酸反应
ARE	+	AAACCA	厌氧诱导所必需的顺式调节元件
AT~TATA-box	+/-	TATATA/TATATAAA
Box4	-	ATTAAT	参与光反应的保守DNA模块的一部分
CAAT-box	+/-	CAAT/CAAAT/CCAAT	启动子和增强子区域的共同顺式作用元件
CAG-motif	-	GAAAGGCAGAC	光响应元件的一部分
CCGTCCmotif	-	CCGTCC
CCGTCC-box	-	CCGTCC
ERE	+/-	ATTTTAAA
G-Box	+	CACGTT	顺式调节元件参与光反应
G-box	-	CACGAC	顺式调节元件参与光反应
GT1-motif	+/-	GGTTAAT/GGTTAA	光响应原件
MBS	+	CAACTG	与干旱诱导有关的MYB结合位点
MYB	+/-	TAACCA/CAACCA
MYB-likesequence	+/-	TAACCA
MYC	+/-	CATGTG
Myb	+	CAACTG
Myc	+	TCTCTTA
TATA	+	TATAAAAT
TATA-box	+/-	TATATA/TATA/ATATAT/TACAAAA	转录开始的-30左右的核心启动子元件
TC-rich repeats	-	GTTTTCTTAC	参与防御和胁迫反应的顺式作用元件
TCT-motif	-	TCTTAC	光响应元件的一部分
Unnamed_1	+	CGTGG
Unnamed_4	+/-	CTTC
Unnamed_6	+	taTAAATATct
Wbox	+	TTGACC
WRE3	-	CCACCT

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黄瓜CsGDSL脂解酶基因的克隆与表达分析

Cloning and Expression Analysis of CsGDSL Lipase Gene in Cucumber

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