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作物学报 ›› 2021, Vol. 47 ›› Issue (5): 882-893.doi: 10.3724/SP.J.1006.2021.04128

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗β-胡萝卜素异构酶基因家族的鉴定、定位和表达分析

黄宁2,*(), 惠乾龙1, 方振名2, 李姗姗2, 凌辉2, 阙友雄1, 袁照年1,*()   

  1. 1福建农林大学国家甘蔗工程技术研究中心, 福建福州 350002
    2玉林师范学院农学院, 广西玉林 537000
  • 收稿日期:2020-06-15 接受日期:2020-09-13 出版日期:2021-05-12 网络出版日期:2020-09-30
  • 通讯作者: 黄宁,袁照年
  • 基金资助:
    国家现代农业产业技术体系建设专项(CARS-170208)

Identification, localization and expression analysis of beta-carotene isomerase gene family in sugarcane

HUANG Ning2,*(), HUI Qian-Long1, FANG Zhen-Ming2, LI Shan-Shan2, LING Hui2, QUE You-Xiong1, YUAN Zhao-Nian1,*()   

  1. 1National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    2College of Crop Science, Yulin Normal University, Yulin 537000, Guangxi, China
  • Received:2020-06-15 Accepted:2020-09-13 Published:2021-05-12 Published online:2020-09-30
  • Contact: HUANG Ning,YUAN Zhao-Nian
  • Supported by:
    China Agriculture Research System(CARS-170208)

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摘要：

独脚金内酯(strigolactones, SLs)是一种广泛存在、能够抑制植物分蘖或分枝的植物激素。β-胡萝卜素异构酶(D27)是SLs合成中的关键酶, 但是目前关于甘蔗D27基因家族的鉴定与分析鲜有报道。本研究通过挖掘甘蔗原始亲本之一的割手密种基因组数据鉴定了5个割手密种D27基因家族成员。系统进化树分析发现, 割手密种D27s分处在3个不同系统发育分支, 与高粱D27s高度同源。保守结构域预测揭示, 割手密种D27s包含β-胡萝卜素异构酶的典型结构域Pfam: DUF4033。顺式元件分析结果显示, 割手密种D27s主要参与调控激素和胁迫响应, 以及植物生长发育等。基于甘蔗栽培种转录组数据分析发现, 甘蔗割手密种D27基因家族成员(Sspon.06G0012830-1A)的同源基因同时参与甘蔗分蘖调控及黑穗病菌胁迫响应。在此基础上, 我们克隆获得了甘蔗栽培种ROC22中的同源基因cDNA序列, 命名为ScD27.1 (GenBank登录号为MT499895)。生物信息学分析结果显示, ScD27.1编码266个氨基酸, 其蛋白等电点为8.91, 分子量为30.00 kD, 是不稳定蛋白且定位于叶绿体。二级结构主要包括α-螺旋和无规则卷曲, 具有叶绿体转运肽, 包含4个泛素化位点和18个磷酸化位点。qRT-PCR表达分析表明, ScD27.1基因受ABA及H2O2显著诱导表达, 但对MeJA、SA胁迫响应不明显。亚细胞定位结果显示, ScD27.1蛋白可能定位于细胞膜和叶绿体, 参与细胞内膜泡运输或由液泡前体、胞内运输小泡分拣运输。以上研究表明, ScD27.1基因可能参与黑穗病菌侵染诱导的甘蔗分蘖及ABA和H2O2相关信号通路。本研究为了解甘蔗ScD27.1蛋白在胞内运输和分蘖中的作用及其参与甘蔗-黑穗病菌互作提供一定的基础理论。

关键词: β-胡萝卜素异构酶, 基因家族, 生物信息学, 分蘖, 甘蔗黑穗病菌

Abstract:

Strigolactones (SLs), a class of plant hormones, exists widely in plants and involves in the regulation of plant tillering and environmental adaptation. β-carotene isomerase (D27) is a key enzyme for SLs synthesis, but there are few reports about the identification and analysis of D27 gene family in sugarcane. In this study, five members of D27 gene family from one of the original parent of modern sugarcane cultivars, Saccharum spontaneum, were analyzed. Phylogenetic tree analysis showed that these D27s from S. spontaneum was clustered into 3 different branches and were highly homologous to sorghum D27s. Conserved domain prediction revealed that D27s contained a typical domain of β-carotene isomerase, Pfam: DUF4033. The results of cis-element analysis showed that D27s mainly involved in the regulation of hormone response, plant growth and development, and stress response. Based on the transcriptomic data of the modern cultivated varieties, the expression analysis of the homologous transcripts of Saccharum spontaneum D27s showed that Sspon.06G0012830-1A was regulated at tillering stage and under Sporisorium scitamineum infection. Besides, the cDNA sequence of Sspon.06G0012830-1A was cloned from sugarcane cultivar ROC22 and named as ScD27.1 (GenBank accession number: MT499895). Bioinformatics analysis indicated that ScD27.1 encoded an unstable protein of 266 amino acids with an isoelectric point of 8.91 and a molecular weight of 30.00 kD, and might be located in the chloroplast, containing chloroplast transit peptides, 4 ubiquitination sites and 18 phosphorylation sites. Its secondary structure mainly included alpha helix and random coils. qRT-PCR analysis demonstrated that ScD27.1 was significantly induced by ABA and H2O2 and did not respond to MeJA and SA. Subcellular localization revealed that ScD27.1 might be located in cell membrane and chloroplast and involved in the vesicle sorting or transported by sorting vesicle in plant cells. The results suggested that ScD27.1 may involve in the tillering induced by S. scitamineum and signaling pathway of ABA and H2O2. The present study provides a basic understanding of intracellular transport and tillering in sugarcane and the involvement of ScD27.1 during sugarcane-S. scitamineum interaction.

Key words: β-carotene isomerase, gene family, bioinformatics, tillering, Sporisorium scitamineum

表1

引物列表"

引物名称 Primer name 引物序列 Primer sequence (5°-3°)
ScD27.1-F CTCGCAGCCTCACAAACGCGGGCGG
ScD27.1-R TCTCAAGCCAACGGTCTCCTA
G-ScD27.1-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGTGCTTCCGCGACC
G-ScD27.1-R GGGGACCACTTTGTACAAGAAAGCTGGGTCCGCCTGAAGTTTGGGACAGA
q-ScD27.1-F GGGTGCTCAAGAAAT
q-ScD27.1-R CTCGTTGGTGAAGAAGTC
CAC-F ACAACGTCAGGCAAAGCAAA
CAC-R AGATCAACTCCACCTCTGCG
CUL-F TGCTGAATGTGTTGAGCAGC
CUL-R TTGTCGCGCTCCAAGTAGTC

图1

甘蔗割手密种D27基因家族鉴定 A: 甘蔗割手密种D27s染色体分布; B: 甘蔗割手密种D27s氨基酸序列间相似性。"

图2

基于甘蔗割手密种和其他5个物种D27s氨基酸序列的系统进化树及结构域分析 横坐标表示蛋白氨基酸序列的长度; 线条颜色表示所用到的物种(Species); 条码颜色表示蛋白的结构域(Domains)。"

图3

甘蔗割手密种D27s顺式元件预测 横坐标表示的基因启动子区核苷酸长度; 条码颜色表示启动子区不同的顺式元件。"

表2

甘蔗割手密种与栽培种D27s间序列相似性"

基因名称
Gene ID		 6-BA和DA-6共同诱导甘蔗栽培种ROC22
分蘖转录组
Transcriptome of sugarcane cultivar ROC22
treated with 6-BA and DA-6		 甘蔗栽培种ROC22和YC05-179应答黑穗病菌
侵染转录组
Transcriptome of sugarcane cultivar ROC22 and YC05-179 responding to Sporisorium scitamineum infection
c116894.
graph_c0		 c112688.
graph_c0		 c161113.
graph_c0		 Sugarcane_
Unigene.66941		 Sugarcane_
Unigene.58014		 Sugarcane_ Unigene.87626
Sspon.06G0012830-3C 94.57 94.96
Sspon.06G0012830-1A 97.93 98.34
Sspon.07G0014180-2B 82.33 98.31
Sspon.07G0014180-3C 80 82.42
Sspon.06G0016140-2C 97.06 95.83

图4

不同转录组中甘蔗栽培种D27s基因的表达 A: 表达数据来源于6-BA和DA-6共同诱导甘蔗栽培种ROC22分蘖相关转录组; B: 表达数据来源于甘蔗栽培种ROC22和YC05-179应答黑穗病菌侵染转录组。X轴表示出现在甘蔗转录组数据中的转录本序号; Y轴表示相对于对照组而言, 转录本在处理组中的相对表达量, 其中*代表相对表达量显著差异(P < 0.05)。"

图5

甘蔗栽培种ScD27.1基因的克隆与测序 A: ScD27.1基因的RT-PCR产物的琼脂糖电泳分析; M代表marker 100 bp-III DNA marker (Generay, 中国上海)。B: ScD27.1基因的开放读码框(*, 终止密码子)及其编码氨基酸序列, 粗体部分为特异性引物在基因序列中的位置。"

表3

生物信息学分析结果"

生物信息学分析 Bioinformatics analysis 结果Result
一级结构特性
Primary structure characteristics		 氨基酸长度 Length of amino acids 266
等电点 Theoretical pI 8.91
分子量 Molecular weight (kD) 30.00
不稳定系数 Instability index (II) 56.40
二级结构类型
Secondary structure types		 α-螺旋 Alpha helix 30.08%
延伸链 Extended strand 12.41%
无规则卷曲 Random coil 57.52%
蛋白亚细胞定位
Protein subcellular location		 Plant-Ploc工具 Plant-Ploc tool 叶绿体Chloroplast
ProtComp 9.0工具 ProtComp 9.0 tool 膜结合叶绿体Membrane bound Chloroplast
WoLFPSORT工具 WoLFPSORT tool 叶绿体Chloroplast
功能位点
Function sites		 靶向肽类型 Type of targeting peptide 叶绿体转运肽Chloroplast transit peptide
泛素化位点 Ubiquitination sites 4
磷酸化位点 Phosphorylation sites 18

图6

ScD27.1基因在不同胁迫下的表达 A: ScD27.1基因定量引物扩增效率曲线; B: ScD27.1基因在ABA、MeJA、SA及H2O2胁迫下的表达。*表示在0.05水平差异显著; 误差线代表各组的标准误差(n = 3)。"

图7

ScD27.1蛋白的亚细胞定位鉴定 A~F为自然状态下蛋白亚细胞定位, 标尺为10 μm; G~L为渥曼青霉素(wortmannin, 16.5 μmol L-1)处理后的蛋白亚细胞定位, 标尺为15 μm。"

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