两种花色绿绒蒿<i>MiUGT79B1</i>和<i>MpUGT79B1</i>基因的克隆及表达分析

doi:10.7668/hbnxb.20194846

摘要/Abstract

摘要：

为探究尿苷二磷酸糖基转移酶(UGT)在不同花色绿绒蒿中的序列特征及其与绿绒蒿花色的关系,以黄色花的全缘叶绿绒蒿和红色花的红花绿绒蒿为研究材料,从2种花色绿绒蒿的全长转录组数据中各筛选1个UGT基因,根据转录组KEGG注释结果,将这2个基因分别命名为MiUGT79B1和MpUGT79B1,并对其进行克隆、生物信息学及RT-qPCR分析。结果表明:MiUGT79B1和MpUGT79B1基因ORF长度为1 314,编码437个氨基酸。MiUGT79B1和MpUGT79B1蛋白的结构均主要由α-螺旋和无规则卷曲构成,属于亲水性稳定蛋白。系统进化树和多序列比对分析发现,MiUGT79B1和MpUGT79B1与鸦片罂粟、火罂粟、野罂粟和博落回的亲缘关系最近。RT-qPCR分析显示,MiUGT79B1和MpUGT79B1均在花蕾期高表达,MiUGT79B1表达趋势为先降低后升高,MpUGT79B1表达趋势为先降低后保持平缓。2种花色绿绒蒿UGT基因表达水平与总黄酮累积模式均呈显著正相关。结果为进一步深入研究MiUGT79B1和MpUGT79B1基因在绿绒蒿花瓣呈色中的功能及其花色分子育种提供理论基础。

关键词: 全缘叶绿绒蒿, 红花绿绒蒿, UGT79B, 基因克隆, 花色

Abstract:

To investigate the sequence characteristics of uridine diphosphate glycosyltransferase in different flower color of Meconopsis and its relationship with the flower color of Meconopsis.The yellow-flowered Meconopsis integrifolia and the red-flowered M.punicea were used as study materials,and one UGT gene was screened from the full-length transcriptome data of each of the two flower colors of Meconopsis.Based on the results of KEGG annotations of the transcriptomes,these two genes were named MiUGT79B1 and MpUGT79B1,and they were cloned,and analyzed by bioinformatic and RT-qPCR.The results showed that the ORF length of MiUGT79B1 and MpUGT79B1 genes was 1 314,encoding 437 amino acids.Both MiUGT79B1 and MpUGT79B1 proteins were hydrophilic stabilizing proteins with their structures consisting mainly of α-helices and random coil.Phylogenetic tree and multiple sequence comparison analyses revealed that MiUGT79B1 and MpUGT79B1 were most closely related to Papaver somniferum,P.californicum, P.nudicaule,and Macleaya cordata.RT-qPCR analysis showed that MiUGT79B1 and MpUGT79B1 were highly expressed in the bud stage,MiUGT79B1 expression tended to decrease first and then increase,and MpUGT79B1 expression tended to decrease first and then remain stable.There was a significant positive correlation between the expression level of UGT gene and the accumulation pattern of total flavonoids.The results of this study provide a theoretical basis for further in-depth research on the function of MiUGT79B1 and MpUGT79B1 genes in the petal coloration of Meconopsis and its molecular breeding of flower color.

Key words: Meconopsis integrifolia, Meconopsis punicea, UGT79B, Gene cloning, Flower color

周麟, 李拓键, 屈燕. 两种花色绿绒蒿MiUGT79B1和MpUGT79B1基因的克隆及表达分析[J]. 华北农学报, 2024, 39(4): 102-109. doi: 10.7668/hbnxb.20194846.

ZHOU Lin, LI Tuojian, QU Yan. Cloning and Expression Analysis of MiUGT79B1 and MpUGT79B1 Genes from Two Varieties of Meconopsis[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(4): 102-109. doi: 10.7668/hbnxb.20194846.

http://www.hbnxb.net/CN/Y2024/V39/I4/102

图/表 13

图1 不同采样时期的全缘叶绿绒蒿(上)和红花绿绒蒿(下)

Fig.1 M.integrifolia(top)and M.punicea(bottom)in different sampling periods

表1 引物序列

Tab.1 Primer sequence

引物名称 Primer name	引物序列(5'—3') Primer sequence (5'—3')
MiUGT79B1-F	ATGGGTCATCTAACTGCTTTCC
MitUGT79B1-R	CTATAGCAGATCCTTAAGCTTGGTG
qMiUGT79B1-F	CTTAGCCACCCTTCCATCGG
qMitUGT79B1-R	CCGCTGTTTTCGTCCATCAC
MpUGT79B1-F	ATGGGTCATCTAACTGCTTTCC
MpUGT79B1-R	CTATAGCAGATCCTGAAGCTTGG
qMpUGT79B1-F	CACCCTTCCATCGGGTGTTT
qMpUGT79B1-R	TTCGTTAGCAACTCCGCCAT
Action-F	TTCCGTGTTCCTACCGTTGA
Action-R	TCCCTTCATCTTACCCTCGG

图2 总RNA电泳检测 M.Marker;1.全缘绿绒蒿总RNA;2.红花绿绒蒿总RNA。

Fig.2 Electrophoresis of total RNA M.Marker;1.Total RNA of M.integrifolia;2.Total RNA of M.punicea.

图3 MiUGT79B1和MpUGT79B1基因电泳 M.Marker;1.MiUGT79B1基因;2.MpUGT79B1基因。

Fig.3 Electrophoresis of MiUGT79B1 and MpUGT79B1 genes M.Marker;1.MiUGT79B1 gene;2.MpUGT79B1 gene.

图4 MiUGT79B1和MpUGT79B1蛋白疏水性分析 A.MiUGT79B1蛋白;B.MpUGT79B1蛋白。图5—8同。

Fig.4 Hydrophobicity analysis of MiUGT79B1 and MpUGT79B1 protein A.MiUGT79B1 protein;B.MpUGT79B1 protein.The same as Fig.5—8.

图5 MiUGT79B1和MpUGT79B1蛋白跨膜结构预测

Fig.5 Transmembrane structure prediction of MiUGT79B1 and MpUGT79B1 protein

图6 MiUGT79B1和MpUGT79B1磷酸化位点预测

Fig.6 Prediction of MiUGT79B1 and MpUGT79B1 phosphorylation sites

图7 MiUGT79B1和MpUGT79B1蛋白二级结构预测蓝线.α-螺旋;红线.延伸链;绿线.β-折叠;紫线.无规则卷曲。

Fig.7 Prediction of the secondary structure of MiUGT79B1 and MpUGT79B1 Blue line.Alpha helix;Red line.Extended strand;Green line.β-sheet;Purple line.Random coil.

图8 MiUGT79B1和MpUGT79B1蛋白三维结构预测

Fig.8 Prediction of the three dimensional structure of MiUGT79B1 and MpUGT79B1

图9 MiUGT79B1和MpUGT79B1系统发育树黑线.同属;红线.同科;蓝线.同目。

Fig.9 Phylogenetic trees of MiUGT79B1 and MpUGT79B1 Black line.Same Genus;Red line.Same family;Blue line.Same order.

图10 MiUGT79B1和MpUGT79B1氨基酸序列比对结果

Fig.10 Results of amino acid sequence alignment between MiUGT79B1 and MpUGT79B1

图11 MiUGT79B1和MpUGT79B1基因表达分析 n=3,不同的小写字母代表显著差异(P<0.05)。图12同。

Fig.11 Expression analysis of MiUGT79B1 and MpUGT79B1 genes n=3,the different lowercase letters represent significant differences(P<0.05).The same as Fig.12.

图12 2种花色绿绒蒿不同时期总黄酮的表达

Fig.12 Expression of total flavonoids of two varieties of Meconopsis in different periods

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两种花色绿绒蒿MiUGT79B1和MpUGT79B1基因的克隆及表达分析

Cloning and Expression Analysis of MiUGT79B1 and MpUGT79B1 Genes from Two Varieties of Meconopsis

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两种花色绿绒蒿MiUGT79B1和MpUGT79B1基因的克隆及表达分析

Cloning and Expression Analysis of MiUGT79B1 and MpUGT79B1 Genes from Two Varieties of Meconopsis

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