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作物学报 ›› 2023, Vol. 49 ›› Issue (2): 414-425.doi: 10.3724/SP.J.1006.2023.24022

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗PIN-LIKES基因家族的鉴定与表达分析

潘洁明1(), 田绍锐3, 梁艳兰2, 朱宇林1, 周定港4, 阙友雄2, 凌辉1,*(), 黄宁1,*()   

  1. 1玉林师范学院农学院, 广西玉林 537000
    2福建农林大学 / 农业农村部福建甘蔗生物学与遗传育种重点实验室, 福建福州 350002
    3西南大学植物保护学院, 重庆 400700
    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:2022-01-17 接受日期:2022-05-05 出版日期:2022-05-12 网络出版日期:2022-05-12
  • 通讯作者: 凌辉,黄宁
  • 作者简介:E-mail: jiemingpan@163.com
  • 基金资助:
    国家自然科学基金项目(31801424);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(G2020ZK03)

Identification and expression analysis of PIN-LIKES gene family in sugarcane

PAN Jie-Ming1(), TIAN Shao-Rui3, LIANG Yan-Lan2, ZHU Yu-Lin1, ZHOU Ding-Gang4, QUE You-Xiong2, LING Hui1,*(), HUANG Ning1,*()   

  1. 1College of Agriculture, Yulin Normal University, Yulin 537000, Guangxi, China
    2Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture and Rural Affairs / Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    3College of Plant Protection, Southwest University, Chongqing 400700, 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:2022-01-17 Accepted:2022-05-05 Published:2022-05-12 Published online:2022-05-12
  • Contact: LING Hui,HUANG Ning
  • Supported by:
    National Natural Science Foundation of China(31801424);National Natural Science Foundation of China(32160435);Scientific Research Foundation of Yulin Normal University(G2020ZK03)

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摘要：

PILS (PIN-LIKES)是一类新发现的生长素输出载体, 协助生长素的极性运输。本研究立足甘蔗割手密基因组和栽培品种转录组数据, 利用生物信息学分析技术, 分别在割手密和栽培品种中鉴定到11个PILS (Saccharum spontaneum PIN-LIKES, SsPILS )基因和4个PILS (Saccharum spp. hybrid PIN-LIKES, ScPILS)基因。结果表明, 11个SsPILS基因家族成员分布于6条染色体上, 基因内含子数量介于5~11个。系统进化树分析发现, 割手密PILS与水稻PILS有较高同源性, 归属于3个不同的系统发育分支。转录组数据分析显示, SsPILS1c的同源基因ScPILS1c在受高粱花叶病毒和黑穗病菌胁迫甘蔗栽培种中差异表达。通过RT-PCR扩增技术, 克隆获得ScPILS1c基因序列(NCBI accession number: OM258732)。该基因全长cDNA为1332 bp, 包含一个1233 bp的完整开放阅读框, 编码410个氨基酸, 理论等电点(pI)为6.17, 不稳定系数为39.31, 平均疏水性值为0.689, 预测ScPILS1c为稳定酸性疏水蛋白。其编码蛋白的二级结构主要包括α-螺旋(48.54%)和无规则卷曲(35.37%), 这与三级结构预测结果相符。qRT-PCR表达分析揭示, ScPILS1c基因在甘蔗中的表达具有组织特异性, 在蔗髓中表达量最低、皮中的表达量最高, 同时该基因的表达受H2O2及黑穗病菌的显著性诱导。亚细胞定位表明, ScPILS1c基因的编码蛋白主要定位于细胞膜。以上结果为深入研究甘蔗PIN-LIKES基因的结构和功能积累了基础资料。

关键词: 甘蔗, PILS, 生物信息学, 实时荧光定量PCR, 基因家族

Abstract:

PILS (PIN-LIKES) is a newly reported auxin efflux carrier, which is involved in the polar transport of auxin. In this study, bioinformatics analysis indicated that 11 SsPILS genes and 4 ScPILS genes were identified from the Saccharum spontaneum genomic and the sugarcane cultivars transcriptomic data. 11 SsPILS gene family members were located at 6 chromosomes, and had 5-11 introns, respectively. Phylogenetic tree analysis showed that these PILS from S. spontaneum was clustered into 3 different branches and had highly homologous with PILS from Oryza sativa. The transcriptomic data revealed that, ScPILS1c, the ortholog of SsPILS1c, was differentially expressed in the sugarcane cultivars under Sorghum mosaic virus and Sporisorium scitamineum stress. ScPILS1c (NCBI accession number: OM258732), cloned by RT-PCR, had 1332 bp in length, containing a 1233 bp complete open reading frame and encoding 410 amino acids residues. The isoelectric point, instability coefficient, and average hydrophobicity value of ScPILS1c were 6.17, 39.31, and 0.689, respectively. The ScPILS1c was predicted as a stable acid hydrophobicity protein. The secondary structure of ScPILS1c protein was mainly composed of α-helix (48.54%) and random coils (35.37%) and was highly consistent with the prediction of tertiary structure. The qRT-PCR demonstrated that the relative expression of ScPILS1c was tissue-specific and the highest expression was observed in epidermis while the lowest in pith. ScPILS1c was significantly induced by H2O2 treatment and Sporisorium scitamineum infection. Transient expression on Nicotiana benthamiana leaves suggested that ScPILS1c was localized on cell membrane. This study provides a reference for further research on the structure and function of PILS gene in sugarcane.

Key words: sugarcane, PILS gene, bioinformatics, qRT-PCR, gene family

表1

本研究所用引物"

引物
Primer name		 引物序列
Primer sequence (5'-3')
PILS1c-F GCGCGGTTGTTGAGGATAAG
PILS1c-R ACGACAATACAACACTTCGGC
YFP-PILS1c-F CGAACGATACTCGAGGTCGACATGGATCTCGTACAACTCTTCATC
YFP-PILS1c-R GCTCACCATACTAGTGGATCCCGACAGCGTCCACATGAAGAAG
q-PILS1c-F AGCTCTTTGCTCCTTCGACC
q-PILS1c-R GACAACTATGACGCCGGCTA
CUL-F TGCTGAATGTGTTGAGCAGC
CUL-R TTGTCGCGCTCCAAGTAGTC
CAC-F ACAACGTCAGGCAAAGCAAA
CAC-R AGATCAACTCCACCTCTGCG

图1

甘蔗割手密PILS基因家族的染色体分布(A)及共线性分析(B) 红色连线表示甘蔗割手密PILS基因家族间的共线性, 灰色连线表示割手密所有基因间的共线性。"

表2

甘蔗割手密PILS蛋白的理化性质分析及亚细胞定位预测"

蛋白名称Protein name 基因编号
Genetic code		 氨基酸个数Number of amino acids 等电点
pI		 分子量
Molecular weight (kD)		 亚细胞定位
Subcellular localization
SsPILS1a Sspon.02G0011550-3C 847 9.76 89.11 质膜 Plasma membrane
SsPILS1b Sspon.02G0011610-1A 436 7.47 46.48 质膜 Plasma membrane
SsPILS1c Sspon.02G0011610-2B 422 7.63 45.10 质膜 Plasma membrane
SsPILS1d Sspon.02G0011610-3C 436 8.02 46.47 质膜 Plasma membrane
SsPILS1e Sspon.02G0011550-1A 412 8.02 44.08 质膜 Plasma membrane
SsPILS5a Sspon.01G0050600-1C 339 7.59 36.61 质膜 Plasma membrane
SsPILS5b Sspon.01G0001780-4D 431 7.01 47.33 质膜 Plasma membrane
SsPILS6a Sspon.03G0003950-1P 433 8.52 46.92 质膜 Plasma membrane
SsPILS6b Sspon.03G0003950-2C 433 8.56 46.97 质膜 Plasma membrane
SsPILS7a Sspon.02G0008260-1A 337 8.10 36.37 质膜 Plasma membrane
SsPILS7b Sspon.02G0008260-2B 301 6.12 32.59 质膜 Plasma membrane

图2

甘蔗割手密(Ss)、拟南芥(At)和水稻(Os) PILS蛋白的系统进化树分析"

图3

甘蔗割手密PILS基因家族蛋白保守基序(A)和基因结构分析(B) A图中, 不同颜色方框表示不同保守基序, 黑线表示氨基酸序列。B图中, 不同颜色方框表示不同基因结构, 黑线表示内含子。"

图4

ScPILS基因在不同胁迫下的表达 A: 高粱花叶病毒胁迫; B: 黑穗病菌胁迫。"

图5

ScPILS1c蛋白生物信息学分析 A: SsPILS1c基因的开放读码框及其所编码蛋白; B: ScPILS1c蛋白的保守结构域; C: ScPILS1c蛋白的跨膜结构; D: ScPILS1c三级结构模型与5aymA 比对; E: ScPILS1c三级结构模型, 红色为跨膜结构域。"

图6

ScPILS1c蛋白氨基酸疏水性/亲水性预测"

图7

ScPILS1c基因在不同组织中的表达 柱上不同小写字母表示在0.05水平差异显著。"

图8

ScPILS1c基因在不同胁迫下的表达 A: H2O2胁迫; B: 黑穗病菌胁迫。柱上不同小写字母表示在0.05水平差异显著。"

图9

甘蔗ScPILS1c在本氏烟下表皮细胞中的亚细胞定位"

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