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作物学报 ›› 2020, Vol. 46 ›› Issue (12): 1914-1922.doi: 10.3724/SP.J.1006.2020.04006

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

红麻海藻糖生物合成关键酶基因HcTPPJ的克隆及响应逆境的表达分析

李辉1,2(), 李德芳2,*(), 邓勇2, 潘根2, 陈安国2, 赵立宁2, 唐慧娟2   

  1. 1湖南文理学院生命与环境科学学院, 湖南常德 415000
    2中国农业科学院麻类研究所, 湖南长沙 410205
  • 收稿日期:2020-01-10 接受日期:2020-08-19 出版日期:2020-09-08 网络出版日期:2020-11-25
  • 通讯作者: 李德芳
  • 基金资助:
    国家现代农业产业技术体系建设专项-红麻育种项目(CARS-19-E07);中国农业科学院科技创新工程一年生麻类育种项目(ASTIP-IBFC03);湖南省教育厅项目(18C0737);湖南文理学院博士科研启动项目(17BSQD13)

Cloning of the key enzyme gene HcTPPJ in trehalose biosynthesis of kenaf and its expression in response to abiotic stress in kenaf

Hui LI1,2(), De-Fang LI2,*(), Yong DENG2, Gen PAN2, An-Guo CHEN2, Li-Ning ZHAO2, Hui-Juan TANG2   

  1. 1College of Life and Environment Science, Hunan University of Arts and Science, Changde 415000, Hunan, China
    2Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, Hunan, China
  • Received:2020-01-10 Accepted:2020-08-19 Published:2020-09-08 Published online:2020-11-25
  • Contact: De-Fang LI
  • Supported by:
    China Agriculture Research System(CARS-19-E07);Agricultural Science and Technology Innovation Program at the Chinese Academy of Agricultural Science(ASTIP-IBFC03);Hunan Education Department Project(18C0737);Doctoral Research Start-up Project of Hunan University of Arts and Sciences(17BSQD13)

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摘要：

海藻糖生物合成关键酶基因TPP在植物响应各种非生物胁迫过程中具有重要作用。为了明确红麻TPP基因在应对非生物胁迫过程中的作用, 本研究根据转录组CL541.Contig2Unigene序列设计特异性引物, 通过PCR获得红麻TPP基因的cDNA全长序列。生物信息学分析表明, 该基因最大开放读码框为1128 bp, 编码一个含有375个氨基酸的蛋白质。序列一致性分析发现, 该蛋白质氨基酸序列与其他物种TPPJ氨基酸序列的一致性为71.18%, 故将该基因命名HcTPPJ。表达模式分析表明, 该基因在根、茎、叶中均有表达, 在盐、干旱胁迫下, 随着胁迫时间的延长, HcTPPJ表达显著上调, 表明该基因参与红麻的盐、干旱胁迫响应过程。在盐、干旱胁迫下, HcNCED3显著上调表达, 而外源喷施脱落酸时, HcTPPJ表达变化不明显。由此推测, 在红麻响应盐、干旱胁迫过程中, 该基因的表达可能不受脱落酸信号分子的调控。这将为进一步阐明该基因在红麻响应盐、干旱胁迫过程的作用奠定基础。

关键词: 红麻, 盐胁迫, 海藻糖-6-磷酸磷酸酶, 脱落酸

Abstract:

Trehalose biosynthesis key enzyme gene TPP plays an important role in plant response to various abiotic stresses. In this study, in order to clarify the role of TPP gene in response to abiotic stress in kenaf, a specific primer was designed according to the sequence of CL541.contig2unigene, and the full-length cDNA sequence of TPP gene was obtained by PCR. Bioinformatics analysis showed that TPP had an open reading frame (ORF) length of 1128 bp, and encoded a protein containing 375 amino acids. The results of amino acid sequence consistency indicated that the agreement between the amino acid sequence of protein and that of TPPJ from other species was 71.18%, so the gene named as HcTPPJ. HcTPPJ was expressed in roots, stems and leaves. Under salt or drought stress, HcTPPJ was up-regulated significantly with the extension of stress treatment, indicating that the gene was involved in the response process of salt or drought stress in kenaf. Under the same conditions, HcNCED3 and HcAOC was significantly up-regulated, while the change of HcTPPJ expression was not obvious under ABA stress; HcTPPJ was significantly down-regulated, under the stress of MeJA for six hours. Therefore it was speculated that the HcTPPJ gene expression may be not regulated by the signal molecule of ABA, but negatively regulated by methyl jasmonate signal molecule. This study will lay a solid foundation for further elucidating the role of the gene in response to salt and drought stress in kenaf.

Key words: kenaf, salt stress, trehalose-6-phosphate phosphatase, ABA

表1

本研究所用引物"

引物名称
Primer name		 引物序列
Primer sequences (5°-3°)		 引物用途
Primer usage
HcTPPJ-F ATGGTGAGTTTCTTTGAA 基因克隆
HcTPPJ-R TTACATTTTAGATTGCCCT Gene cloning
HcTPPJ-QF AACCTTTCTGCCTTGAGT 实时荧光定量PCR
HcTPPJ-QR AAATTGGCTGAGCTGTAC qRT-PCR
HcNCED3-QR AGGCGGTCGTCGGACTCGTT 实时荧光定量PCR
HcNCED3-QF GACTGCTTCTGCTTCCACCTCTG qRT-PCR
Actin-QF CAGGCAGTTCTTTCTTTGT 内参基因
Actin- QR ATCCTCCAATCCAGACACT Reference gene

图 1

HcTPPJ cDNA全长琼脂糖凝胶电泳 M: DNA marker 2K Plus II; 1: PCR产物。"

图2

红麻HcTPPJ与其他植物TPPJ蛋白氨基酸序列一致性比对 OsTPP1: 水稻; AtTPPA、AtTPPB、AtTPPD: 拟南芥; TPPJ: 可可树、木槿、榴莲、雷蒙德氏棉、毛果杨、红麻、橡胶树、石榴。不同颜色代表不同氨基酸残基的保守性。蓝色表示氨基酸完全保守; 粉红色、青色、黄色分别表示氨基酸的保守性为75%以上、50%以上及33%以上; 白色表示氨基酸的保守性不足33%。"

图3

红麻HcTPPJ基因与其他植物TPPJ基因系统进化树 TPPJ: 可可树、木槿、榴莲、雷蒙德氏棉、毛果杨、红麻、橡胶树、石榴; AtTPPA、AtTPPB、AtTPPD: 拟南芥; OsTPP1: 水稻。"

图4

HcTPPJ基因在不同器官的表达 * 表示在0.05水平下差异显著。误差线为每组处理的标准误差(n = 3)。"

图5

150 mmol L-1 NaCl胁迫3 d后HcTPPJ基因在不同器官的表达 *与**分别表示在0.05和0.01水平下差异显著。误差线为每组处理的标准误差(n = 3)。"

图6

不同NaCl浓度胁迫下HcTPPJ基因在叶片的表达 *与**分别表示在0.05和0.01水平下差异显著。误差线为每组处理的标准误差(n = 3)。"

图7

150 mmol L-1 NaCl胁迫下不同时间HcTPPJ基因在叶片的表达 **表示在0.01水平下差异显著。误差线为每组处理的标准误差(n = 3)。"

图8

干旱胁迫8 h HcTPPJ在不同器官的表达(A)和干旱胁迫下不同时间HcTPPJ基因在叶片的表达(B) *与**分别表示在0.05和0.01水平下差异显著。误差线为每组处理的标准误差(n = 3)。"

图9

盐(A)、干旱(B)胁迫下HcNCED3基因在不同时间的表达 *与**分别表示在0.05和0.01水平下差异显著。误差线为每组处理的标准误差(n = 3)。"

图10

ABA胁迫下HcTPPJ基因在不同时间的表达 误差线为每组处理的标准误差(n = 3)。"

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