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作物学报 ›› 2023, Vol. 49 ›› Issue (7): 1860-1870.doi: 10.3724/SP.J.1006.2023.24259

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

转录组分析揭示光诱导转录因子StMYB113调控马铃薯块茎表皮叶绿素合成

赵喜娟1,2(), 刘圣宣2, 刘腾飞2, 郑洁2, 杜鹃2, 胡新喜1, 宋波涛2,*(), 何长征1,*()   

  1. 1湖南省蔬菜生物学重点实验室 / 园艺作物种质创新与新品种选育研究中心 / 湖南农业大学, 湖南长沙 410128
    2农业农村部马铃薯生物学与生物技术重点实验室 / 园艺植物生物学教育部重点实验室 / 华中农业大学, 湖北武汉 430070
  • 收稿日期:2022-11-21 接受日期:2023-02-24 出版日期:2023-07-12 网络出版日期:2023-03-02
  • 通讯作者: *何长征, E-mail: hecz@hotmail.com; 宋波涛, E-mail: songbotao@mail.hzau.edu.cn
  • 作者简介:E-mail: 704986451@qq.com
  • 基金资助:
    本研究由湖北省第三批现代农业产业技术体系项目(鄂采计[2020]-09533号);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-09-P07)

Transcriptome analysis reveals the regulatory role of the transcription factor StMYB113 in light-induced chlorophyll synthesis of potato tuber epidermis

ZHAO Xi-Juan1,2(), LIU Sheng-Xuan2, LIU Teng-Fei2, ZHENG Jie2, DU Juan2, HU Xin-Xi1, SONG Bo-Tao2,*(), HE Chang-Zheng1,*()   

  1. 1Key Laboratory for Vegetable Biology of Hunan Province / ERC for Germplasm Innovation and New Variety Breeding of Horticultural Crops / Hunan Agricultural University, Changsha 410128, Hunan, China
    2Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs / Key Laboratory of Horticultural Plant Biology, Ministry of Education / Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2022-11-21 Accepted:2023-02-24 Published:2023-07-12 Published online:2023-03-02
  • Contact: *E-mail: hecz@hotmail.com; E-mail: songbotao@mail.hzau.edu.cn
  • Supported by:
    The Third Batch of Modern Agricultural Industrial Technology System Project in Hubei Province(鄂采计[2020]-09533号);The China Agriculture Research System of MOF and MARA(CARS-09-P07)

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摘要：

马铃薯块茎见光变绿严重影响了其食用安全性和经济效益, 但光诱导马铃薯块茎合成叶绿素的机制尚不清楚。本研究对不同光照时间处理的马铃薯块茎进行了相关代谢产物分析, 结果表明随着光照时间的延长块茎叶绿素含量逐渐上升, 且在36 h时叶绿素含量上升显著, 块茎表皮也随之出现明显的绿色; 对光诱导0、6、36 h的样品的转录组测序和生物信息学分析共鉴定到5646个差异表达基因(DEGs), 进一步通过共表达聚类分析和qRT-PCR定量验证, 9个主要的叶绿素生物合成结构基因StGAS1、StCHLD、StCrd1、StHEMA、StGUN4、StPORA、StUROD、StCHLM、StCHLG以及6个转录因子StSBP、StLSD、StGATA、StWRKY、StMYB-like、StMYB113显著上调。对这9个结构基因的启动子序列的顺式作用元件的预测结果显示它们都含有多个MYB结合位点, 启动子元件分析和转录激活验证试验结果表明StMYB113具有光响应元件, 并能在烟草激活StUROD的表达, 由此说明StMYB113可能受光响应且调控马铃薯块茎的见光变绿。本研究结果为光诱导马铃薯块茎叶绿素合成调控机制研究提供参考, 对减少马铃薯块茎绿化造成的损失具有重要意义。

关键词: 马铃薯, 见光绿化, 叶绿素合成, StMYB113, 转录激活

Abstract:

Light-induced greening of tubers seriously affects its safety and economic benefits, but the mechanism of light-induced chlorophyll synthesis in potato tubers remains unclear. In this study, the related metabolites of potato tubers with different light durations were analyzed. The results were as follows: When the light duration prolonged, the chlorophyll content of the tubers gradually increased. Moreover, the chlorophyll content increased significantly at 36 hours, with which the tuber skin turned green obviously. Transcriptome sequencing and bioinformatics analysis were carried out on the samples taken at 0 hour, 6 hours, and 36 hours, and 5646 differentially expressed genes (DEGs) were identified. According to the co-expression cluster analysis and quantitative RT-PCR verification results, 9 major structure genes of chlorophyll biosynthesis pathway (StGAS1, StCHLD, StCrd1, StHEMA, StGUN4, StPORA, StUROD, StCHLM, and StCHLG) and 6 transcription factors (StSBP, StLSD, StGATA, StWRKY, StMYB-like, and StMYB113) were significant induced. Predictions of the cis-acting elements of the promoter sequences of these 9 structural genes indicated that they all contained multiple MYB binding sites. Promoter element analysis and transcriptional activation validation further revealed that StMYB113 had a light-responsive element and could activate the expression of StUROD in tobacco. Therefore, StMYB113 may be light-responsive and be able to regulate potato tuber greening in the light. This study provides a reference for the research on the regulatory mechanism of light-induced chlorophyll synthesis in potato tubers, which is important for reducing the loss caused by greening of potato tubers.

Key words: potato, light-induced greening, chlorophyll synthesis, StMYB113, transcriptional activation

表1

定量引物序列"

基因名称 正向引物 反向引物 基因ID
Gene name Forward primer (5°-3°) Reverse primer (5°-3°) Gene ID
GSA1 GTGGTGGGTTCATAAGGGGG TGGAGTGTGTGCTAAGCTGG Soltu.DM.04G005660.2
CHLD GACGCTAGTGGGAGTATGGC CCCCACCACAGGGAAGTTTT Soltu.DM.04G010510.1
Crd1 TGGTGCCAAGATGAGAACCG TGTGACATAAACCGAAAGGCA Soltu.DM.10G030260.1
HEMA GAGTGATTCATGGGAGAGATCCA GCAGGGTTCCAAAGTGAATGA Soltu.DM.04G031570.1
GUN4 TCGTTTTAGCCGGAGAAGCA TAACCGAATTTGCCGTCGCT Soltu.DM.06G028640.1
PORA GAGGGCAAAGCTAGTGCAAC TGACACCAGGAGAAGCAACC Soltu.DM.10G002270.1
UROD AGCTAGGCTTTCTCATTGTGT AAGCAAACCACAGAGACGGA Soltu.DM.06G011840.1
CHLM AACGCTAACGAATGGCAACG CGGCGAGGAGAAAGCCATAA Soltu.DM.03G032490.1
CHLG ACAACAACCCACTTCGCTCT TTATCCGGTGCCTGTGCTTT Soltu.DM.09G000240.1
SBP TCTGTCAACAATGCAGCCGA CTTCGCCTTTCGTTGTGTCC Soltu.DM.07G020030.1
LSD GCAGTAACAGCAGTACCACCT CACGAACATTGCACGCTTGT Soltu.DM.08G022900.1
GATA CAGCAGCGGAGAAAGTACGA GCCATCAACAAGAATGCTGCT Soltu.DM.12G001590.1
WRKY ACAGGGGTGAACACAACCAC GTCATTGCATACGGCTGCTC Soltu.DM.10G023760.1
MYB-like AAGACCTCAACCTCGGAACT TGCAACGTTTGTCGTCTTTGT Soltu.DM.10G020840.1
MYB113 TGGAACACACACCTACACAGG TCGAGGTCGTGGTCGTAGTA Soltu.DM.10G020780.1

图1

RM-210块茎在光照条件下0~120 h的表型和叶绿素含量变化 (A)光照0、6、12、36、48、120 h后块茎图片; (B)光照120 h期间叶绿素a和叶绿素b的含量变化。数据为3个重复的平均值±标准偏差。不同字母表示由方差分析确定的各组间的统计学差异(Tukey多重比较, P < 0.05)。"

图2

光照0、6和36 h块茎中DEGs的聚类图和富集分析 (A) 转录组数据的主成分分析; (B) 6 h vs 0 h和36 h vs 0 h的DEG火山图。蓝色代表显著表达的基因, 灰色代表不显著表达的基因; (C) DEGs的GO富集分析; (D) DEGs的KEGG通路分析。"

图3

光照0、6、36 h的块茎中DEG的聚类分析"

图4

qPCR测定RM-210块茎中叶绿素合成相关基因的表达 (A): 叶绿素生物合成途径中的结构基因; (B): 提取22℃下光照0、6、36和48 h 时块茎皮中的RNA, qPCR技术检测9个结构基因的表达量。不同字母表示由方差分析确定的各组间的统计学差异(Tukey多重比较, P < 0.05)。"

图5

qPCR验证转录因子表达量 (A): 转录因子的qPCR结果; (B): 转录因子的RNA-seq数据与qPCR数据的比较; (C): 转录因子的RNA-seq数据与qPCR数据的相关性分析(Pearson相关性分析)。不同字母表示由方差分析确定的各组间的统计学差异(Tukey多重比较, P < 0.05)。"

图6

结构基因(A)和转录因子(B)的启动子顺式元件预测"

图7

MYB转录因子对结构基因启动子的转录激活 EV代表空载与结构基因的启动子重组载体共注射烟草; TF代表相应转录因子重组载体与结构基因的启动子重组载体共注射烟草。星号表示由Student’s t测验确定的试验组与空载间的统计学差异(*为P < 0.05, **为P < 0.01)。"

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