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作物学报

作物学报 ›› 2023, Vol. 49 ›› Issue (9): 2485-2497.doi: 10.3724/SP.J.1006.2023.24233

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗ScbHLH13基因的RT-PCR克隆与功能分析

莫广玲1(), 余陈静3, 梁艳兰1, 周定港4, 罗俊1, 王莫1, 阙友雄1, 黄宁2,*(), 凌辉2,*()   

  1. 1福建农林大学国家甘蔗工程技术研究中心 / 福建农林大学生命科学学院, 福建福州 350002
    2玉林师范学院农学院, 广西玉林 537000
    3贵阳市教育科学研究所, 贵州贵阳 550000
    4湖南科技大学生命科学学院 / 经济作物遗传改良与综合利用湖南省重点实验室, 湖南湘潭 411201
  • 收稿日期:2022-10-17 接受日期:2023-02-21 出版日期:2023-09-12 网络出版日期:2023-03-10
  • 通讯作者: *黄宁, E-mail: hning2012@126.com; 凌辉, E-mail: linghuich@163.com
  • 作者简介:莫广玲, E-mail: guanglingmo@163.com
  • 基金资助:
    国家自然科学基金项目(31901592);国家自然科学基金项目(32160435);玉林师范学院高层次人才启动项目(G2022ZK15)

RT-PCR cloning and functional analysis of ScbHLH13 in sugarcane

MO Guang-Ling1(), YU Chen-Jing3, LIANG Yan-Lan1, ZHOU Ding-Gang4, LUO Jun1, WANG Mo1, QUE You-Xiong1, HUANG Ning2,*(), LING Hui2,*()   

  1. 1National Engineering Research Center for Sugarcane / College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    2College of Crop Science, Yulin Normal University, Yulin 537000, Guangxi, China
    3Guiyang Research Institute of Education Science, Guiyang 550000, Guizhou, China
    4College of Life Science, Hunan University of Science and Technology / Key Laboratory of Economic Crops Genetic Improvement and Integrated Utilization, Xiangtan 411201, Hunan, China
  • Received:2022-10-17 Accepted:2023-02-21 Published:2023-09-12 Published online:2023-03-10
  • Supported by:
    National Natural Science Foundation of China(31901592);National Natural Science Foundation of China(32160435);Scientific Research Foundation of Yulin Normal University for High-level Talents(G2022ZK15)

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摘要：

bHLH转录因子是植物体内重要的调节因子, 对植物的生长发育、次生代谢以及花青素生物合成等方面具有调节作用。本研究以高粱bHLH13 (XM_002440799.2)为探针序列, 从甘蔗中通过RT-PCR克隆获得一个ScbHLH13基因, 并对其在不同胁迫响应转录组数据中进行表达模式分析, 同时对该基因及其所编码蛋白进行了理化性质预测以及系统进化、原生质体亚细胞定位和实时荧光定量PCR表达量分析。结果显示, ScbHLH13所编码蛋白包含586个氨基酸残基, 以无规则卷曲和α螺旋为主, 亲水且不稳定, 呈弱碱性; 具有典型核定位信号, 无跨膜结构; 其序列内包括bHLH-MYC、HLH 2个典型保守结构域, 属于bHLH超家族成员。在系统进化中ScbHLH13属于III (d+e)类组, 与高粱亲缘关系最近。在转录组数据中, ScbHLH13的表达在甘蔗品种ROC22上受低氮胁迫和黑穗病菌侵染抑制, 轻微受高粱花叶病毒侵染诱导; 而在Badila受低氮、在YC05-179受甘蔗黑穗病菌侵染诱导。亚细胞定位结果显示, 在烟草表皮细胞中瞬时表达ScbHLH13定位于细胞核和细胞膜, 在甘蔗原生质体瞬时表达ScbHLH13则定位于细胞核。qRT-PCR分析表明, ScbHLH13在甘蔗原生质体中过表达并不能诱导花青素合成代谢通路上主要基因的表达, 说明ScbHLH13与甘蔗中花青素合成相关下游基因表达调控相关性较低。本研究借助甘蔗原生质体瞬时表达系统上对ScbHLH13进行了初步的表达调控探究, 为进一步研究甘蔗功能基因研究以及bHLH基因家族的结构与功能研究奠定了一定的基础。

关键词: 甘蔗, 原生质体, bHLH转录因子, ScbHLH13基因

Abstract:

The transcription factors in bHLH family are important regulators in controlling plant growth and development, secondary metabolism, and anthocyanin biosynthesis. In this study, we successfully cloned ScbHLH13 using sorghum bHLH13 (XM_002440799.2) as the probe sequence by RT-PCR in sugarcane. Meanwhile, the physicochemical properties of the gene and its encoded protein were predicted. Phylogenetic relationship, subcellular localization, and the relative expression pattern of ScbHLH13 were investigated. ScbHLH13 contained 586 amino acids, and mainly consisted of random coils and α-helice. It was hydrophilic, unstable, and weakly basic. Moreover, ScbHLH13 had the typical nuclear localization signals and no transmembrane. The sequence contained two typical conserved domains, bHLH-MYC, and HLH, which belonged to the bHLH superfamily. Analysis of sugarcane transcriptomes under low nitrogen, and Sorghum mosaic virus, and Sporisorium scitamineum invasion revealed that the transcriptional levels of ScbHLH13 were altered in response to various abiotic and biotic stresses. When transiently expressed in tobacco, ScbHLH13-YFP was observed in nuclei and on cell membrane, whereas it specifically localized in nuclei of sugarcane protoplasts. QRT-PCR assays showed that relative expression level of ScbHLH13 in sugarcane protoplasts could not induce transcription of the relative genes in anthocyanin synthesis and the metabolism pathway, suggesting that ScbHLH13 may not involve in anthocyanin biosynthesis and metabolism. In conclusion, this study explores the basic features and functions of ScbHLH13, which will be helpful for the future research on characterizing the biological roles of bHLH members in regulating sugarcane growth and defense.

Key words: sugarcane, protoplast, bHLH transcription factor, ScbHLH13 gene

表1

ScbHLH13基因和花青素分支通路的合成基因的qRT-PCR引物"

引物名称
Primer		 上游引物
Forword primer (5°-3°)		 下游引物
Reverse primer (5°-3°)		 引物用途
Primer function
ScbHLH13 CTAGGTCTTCATCAGTCCTTC TATACCATGTATACCGTCAGTG 基因克隆
Gene cloning
ScbHLH13-Gate GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAAGACAGAGATAGAAGAGGTA GGGGACCACTTTGTACAAGAAAGCTGGGTCCACAGAGCTAGTTGCATTAGAAAT 构建表达载体
Constructing the expression vector
ScbHLH13-Q TCGACTTCCAGAGCCAACAC TTGGCTGTGACCTTCGACTC 实时荧光定量PCR
Real-time quantitative PCR
F3’H-Q GAGCTGCTAGTCAATGTGTGGG AGTTAGTGTGACCATCCGTAGC
A1-Q GATCCTGAAGCAGGTGCAGTTC AACCTCTCCGGGATGTCGT
CHI-Q GAGAAGTTCACAAGGGTGACCA GACGAGTCCTTGGAGAAGGC
F3H-Q GAGCAACGGCAGGTTCAAGA TACATCTCGGCGAAGGTGATG
CHS-Q GTTCCACCTGCTCAAGGACG GACATGTTGCCGTACTCGGAGA
ANS-Q CAGGCTCAGACCAATTGACAAC GACAGAATGAATGCTGGCTGAG
CUL TGCTGAATGTGTTGAGCAGC TTGTCGCGCTCCAAGTAGTC 内参基因
Reference genes
CAC ACAACGTCAGGCAAAGCAAA AGATCAACTCCACCTCTGCG

图1

ScbHLH13的核酸序列及其所编码氨基酸序列特征 A: 高粱bHLH13和ScbHLH13核酸序列比对, 绿色显示非保守性核酸位点; B: ScbHLH13蛋白保守结构域预测; C: ScbHLH13蛋白跨膜结构预测。"

图2

甘蔗ScbHLH13及同源物种bHLH13编码的氨基酸序列的多重比对"

图3

基于AlphaFold建模的bHLH13蛋白三级结构分析 甘蔗(Saccharum spp. hybrids, GenBank登录号为USN24528.1)、高粱(Sorghum bicolor, GenBank登录号为XP_002440844.1)、谷子(Setaria italica, GenBank 登录号为: XP_004960675.1)、黍(Panicum miliaceum, GenBank登录号为RLN28435.1)、大麦(Hordeum vulgare, GenBank登录号为KAE8807589.1)、粗山羊草(Aegilops tauschii, GenBank登录号为XP_020150590.1)的bHLH13蛋白质三维结构模型均来源于AlphaFold数据库(https://alphafold.com/); 蓝色虚线和黑色箭头为各物种bHLH13蛋白质三维结构差异区, 其中蓝色虚线为存在差异的红色β折叠区, 黑色箭头为存在差异的无规卷曲或α螺旋。"

图4

不同植物bHLH蛋白的系统进化树 红色字体为ScbHLH蛋白; 参考前人对bHLH家族的分类[31], 分别从拟南芥和水稻bHLH家族中选取18个代表各亚家族的序列[21,32]构建进化树, 不同颜色框内表示不同bHLH亚家族, 其中包括VII (a+b)、VIIIb、III (a+c)、IIIb、III (d+e)、IIIf。"

图5

不同转录组中ScbHLH13基因表达量 A: 表达数据源于甘蔗品种Badila和ROC22受低氮胁迫转录组; B: 表达数据源于健康和受高粱花叶病毒侵染的甘蔗品种ROC22的转录组; C: 表达数据源于受黑穗病菌胁迫甘蔗品种YC05-179和ROC22的转录组。"

图6

ScbHLH13蛋白在本氏烟叶片表皮的亚细胞定位 图片采用明场、黄色荧光、叠加场3个拍摄通道。YFP是带有空载pEarlegate101-YFP的农杆菌菌液注射结果。ScbHLH13:: YFP是带有重组载体pEarlegate101-ScbHLH13-YFP的农杆菌菌液注射结果。"

图7

ScbHLH13蛋白在甘蔗叶鞘原生质体中的亚细胞定位分析 细胞内是YFP定位。荧光显微镜选取绿色荧光通道, 激发光波长: 358~360 nm, 发射光波长: 460~461 nm。标尺为10 μm。"

图8

甘蔗原生质体中花青素合成基因的表达模式 图中bHLH表示瞬时表达ScbHLH13基因的试验组。F3’H、A1、CHI、F3H、CHS、ANS为花青素合成分支通路成员。bHLH为试验组, YFP为对照组。柱上不同的小写字母代表显著性差异(P < 0.05)。"

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