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作物学报 ›› 2023, Vol. 49 ›› Issue (11): 2978-2990.doi: 10.3724/SP.J.1006.2023.24247

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

马铃薯PAL基因家族的全基因组鉴定及其在非生物胁迫下和块茎花色素苷合成中的表达分析

朱金勇1(), 刘震1, 曾钰婷2, 李志涛1, 陈丽敏1, 李泓阳1, 史田斌1, 张俊莲3, 白江平1, 刘玉汇1,*()   

  1. 1甘肃农业大学农学院 / 省部共建干旱生境作物学国家重点实验室(甘肃农业大学) / 甘肃省作物遗传改良与种质创新重点实验室, 甘肃兰州 730070
    2西藏自治区农牧科学院蔬菜研究所, 西藏拉萨 850032
    3甘肃农业大学园艺学院, 甘肃兰州 730070
  • 收稿日期:2022-11-05 接受日期:2023-04-17 出版日期:2023-11-12 网络出版日期:2023-05-11
  • 通讯作者: 刘玉汇, E-mail: lyhui@gsau.edu.cn
  • 作者简介:E-mail: 1259245907@qq.com
  • 基金资助:
    国家自然科学基金项目(31860398);甘肃省科技计划资助项目(22JR5RA834);财政部和农业农村部国家现代农业产业技术体系资助项目(CARS-09-P14);省部共建干旱生境作物学国家重点实验室(甘肃农业大学)开放基金项目(GSCS-2021-Z02);甘肃农业大学“伏羲人才”计划项目(Gaufx-02Y04);甘肃农业大学公招博士科研启动基金项目(GAU-KYQD-2020-11)

Genome-wide identification of potato (Solanum tuberosum L.) PAL gene family and its expression analysis in abiotic stress and tuber anthocyanin synthesis

ZHU Jin-Yong1(), LIU Zhen1, ZENG Yu-Ting2, LI Zhi-Tao1, CHEN Li-Min1, LI Hong-Yang1, SHI Tian-Bin1, ZHANG Jun-Lian3, BAI Jiang-Ping1, LIU Yu-Hui1,*()   

  1. 1College of Agronomy, Gansu Agricultural University / State Key Laboratory of Aridland Crop Science (Gansu Agricultural University) / Gansu Provincial Key Laboratory of Crop Improvement and Germplasm Enhancement, Lanzhou 730070, Gansu, China
    2Institute of Vegetable Sciences, Tibet Academy of Agriculture and Animal Husbandry Sciences, Lhasa 850032, Tibet, China
    3College of Horticulture, Gansu Agricultural University, Lanzhou 730070, Gansu, China
  • Received:2022-11-05 Accepted:2023-04-17 Published:2023-11-12 Published online:2023-05-11
  • Supported by:
    National Natural Science Foundation of China(31860398);Science and Technology Program of Gansu Province(22JR5RA834);China Agriculture Research System of MOF and MARA(CARS-09-P14);State Key Laboratory of Aridland Crop Science of China(GSCS-2021-Z02);Fuxi Talent Project of Gansu Agricultural University(Gaufx-02Y04);Scientific Research Startup Funds for Openly-recruited Doctors Agricultural University(GAU-KYQD-2020-11)

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摘要：

苯丙氨酸解氨酶(phenylalanin ammonia-lyase, PAL)是苯丙烷代谢途径的限速酶和关键酶, 在植物生长发育过程中发挥着重要作用。本研究在马铃薯(Solanum tuberosum L.)全基因组水平下, 利用BlastP和HMM 3.1鉴定到8个PAL基因家族成员, 利用ExPASy、CELLO、PlantCARE等在线工具, 对PAL基因家族成员的进行生物信息学分析。利用PGSC数据库下载的RNA-seq数据, 分析了StPALs在双单倍体(DM)马铃薯不同组织部位和非生物胁迫下的表达模式。对3个不同颜色马铃薯块茎组织(皮和肉)进行RNA-seq, 以及利用3个不同颜色块茎杂交子代的薯肉进行qPCR分析。结果表明, 8个StPALs分布在3、5、9和10号染色体上, 它们与烟草(Nicotiana tabacum) PAL的亲缘关系较近。StPAL基因成员的启动子区域内含有多个顺式元件, 包括光响应、逆境胁迫响应、激素响应、生长发育相关和转录因子调控的多种顺式元件。StPAL2在匍匐茎中特异表达, StPAL3/5/8在块茎和匍匐茎中特异表达, StPAL3在甘露醇处理下下调表达, StPAL3和StPAL8在热胁迫中上调表达。它们可能参与了马铃薯块茎的发育和非生物胁迫响应。5个StPALs (StPAL3/4/5/6/8)基因在彩色薯肉中均上调表达, 可能参与马铃薯薯肉中花色素苷的生物合成。这些结果为进一步了解StPAL基因家族特征, 深入分析StPALs基因在马铃薯中的功能提供了理论依据。

关键词: 马铃薯, PAL基因家族, 非生物胁迫, 花色素苷生物合成, 表达分析

Abstract:

The phenylalanine ammonia-lyase (PAL) is the rate limiting enzyme and key enzyme in phenylpropane metabolism pathway, which plays an important role in plant growth. In this study, a total of 8 gene family members (StPALs) in potato (Solanum tuberosum L.) were identified by BlastP and Hmmer 3.1 software, and their bioinformation were analyzed by the ExPASy, CELLO, PlantCARE, and other online tool. We analyzed the relative expression pattern of StPAL genes in different tissues of double monoploid (DM) potato, as well as under abiotic stresses by RNA-seq in Potato Genome Sequencing Consortium (PGSC) database. We performed RNA-seq on white, red, and purple tuber skin and flesh of three potato cultivars, and the relative expression levels of StPALs genes in different colors tubers (flesh) of three hybrid progeny potatoes were detected by qPCR. StPAL genes were distributed on chromosomes 3, 5, 9, and 10, and eight StPALs were closely related to tobacco (Nicotiana tabacum) PAL. The cis-acting elements revealed that the promoter regions of StPAL genes contained many elements, including light response, stress response to adversity, hormone response, growth and development, and transcription factor binding elements. The results showed that StPAL2 was specifically expressed in stolons, and StPAL3/5/8 were mainly expressed in tubers and stolons. The relative expression level of StPAL3 gene were down-regulated under mannitol treatment, and the relative expression levels of StPAL3 and StPAL8 genes were up-regulated under heat stress. The above results suggested that StPALs might be involved in the tuber growth and abiotic stress response. By transcriptomic and qPCR analysis, the relative expression levels of 5 StPAL genes (StPAL3/4/5/6/8) were up-regulated in the flesh of color potato, suggesting that they may participate in the biosynthesis of anthocyanins in flesh. These results provide a theoretical basis for further understanding the StPAL gene family and analyzing the function of StPALs in potato.

Key words: potato, PAL genes family, abiotic stress, anthocyanin biosynthesis, expression analysis

图1

3个不同颜色马铃薯品种的块茎[35]"

图2

3个马铃薯杂交子代的块茎表型"

表1

qPCR引物序列"

基因名称
Gene name		 正向引物
Forward primer (5°-3°)		 反向引物
Reverse primer (5°-3°)
StEF-1α GGTCGTGTTGAGACTGGTGTGATC GCTTCGTGGTGCATCTCTACAGAC
PG2021549 CTCACAGCAGGAAGGAATCCAAGC TGAAGTTCCGAGCAGTAAGAAGCC
PG0031457 CTCACAGCAGGAAGGAATCCAAGC GCTCGGCACTCTGAACATGGTTAG
PG0005492 ATCCGACTAGGTGGTGGTGAGATG ATCCAATCACTGCTCGCCTTGAC
PG0023458 CACAGCGTCTGGTGACTTGGTAC GCGTCCAACAGTTCTCCATTAGGC
PG1021549 CTGCTGAGGCTGTGGACATCTTG TTGGCTACTTGGCTTACGGTGTTC
PG2021564 TGGAACGGTCACTGCCTCAGG CACTAACACCAGCCACACGGAAC
PG0019386 ACTGCCTCGGGTGATCTTGTCC ACTAATACCAGCAACACGGAACGC
PG0031365 GCCGAAGGAAGGACTTGCTCTTG CGGGCTTTCCATTCATCACCTCAG

表2

StPAL基因家族理化性质及亚细胞定位"

基因名称
Gene
name		 氨基酸长度
Amino acid
length		 相对分子量
Molecular
weight (kD)		 等电点
Point
isoelectric (pI)		 亚细胞定位
Subcellular
localization		 染色体定位
Chromosome
localization
PG0031457, StPAL1 435 48.34 5.84 细胞质Cytoplasmic Chr03
PG0005492, StPAL2 667 73.69 5.41 细胞质Cytoplasmic Chr05
PG0023458, StPAL3 707 77.51 6.07 细胞质Cytoplasmic Chr05
PG2021549, StPAL4 391 43.55 6.62 细胞质Cytoplasmic Chr09
PG1021549, StPAL5 722 78.49 6.04 细胞质Cytoplasmic Chr09
PG2021564, StPAL6 719 78.23 6.15 细胞质Cytoplasmic Chr09
PG0019386, StPAL7 689 75.46 6.28 叶绿体Chloroplast Chr10
PG0031365, StPAL8 711 77.54 5.86 细胞质Cytoplasmic Chr10

图3

多个物种中PALs蛋白进化树 红色圆表示StPALs, 绿色五角星表示AtPALs, 绿色六边形表示NtPALs。"

图4

StPALs基因家族的进化关系、基因结构和保守基序分析 A: StPALs进化树。B: StPALs基因的外显子/内含子结构。蓝色框表示外显子, 相同长度的黑线表示内含子。上游/下游区域红色方框表示。C: StPALs中保守基序的分布。不同颜色的框代表10个不同的基序。"

图5

马铃薯PAL基因启动子顺式作用元件预测"

图6

StPALs在不同组织部位(A)、非生物胁迫(B)和不同薯皮薯肉(C)中的表达 A: 对8个StPALs基因表达量取以2为底的对数, 用log2FPKM绘制色标。B: 在3种非生物胁迫(盐、甘露醇和热胁迫)中使用处理比对照的比值, 取以2为底的对数, 用log2FC绘制色标。C: 在不同薯皮薯肉中使用彩色品种/白色品种的比值, 取以2为底的对数, 用log2FC绘制色标。XDS、LTS和HMS分别代表“新大坪”、“铃田红美”和“黑美人”的薯皮。XDF、LTF和HMF分别代表“新大坪”、“铃田红美”和“黑美人”的薯肉。"

图7

8个StPAL基因在不同颜色块茎(薯皮和薯肉)中的qPCR分析 对8个StPALs在白色和彩色薯皮和薯肉中的表达进行了qPCR分析。XDS、LTS和HMS分别代表“新大坪”、“红美”和“黑美人”的薯皮。XDF、LTF和HMF分别代表“新大坪”、“铃田红美”和“黑美人”的薯肉。数据为3个独立生物重复的平均值(±SE)。条形图上方的不同字母表示P < 0.05时的显著差异。"

图8

8个StPAL基因在3个马铃薯杂交子代(Y、R、P)薯肉中的qPCR分析 对8个StPALs在Y、R和P薯肉中的表达进行了qPCR分析。数据为3个独立生物重复的平均值(±SE)。条形图上方的不同字母表示P < 0.05时的显著差异。缩写同图2。"

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