欢迎访问作物学报，今天是
作物学报

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)

RichHTML

5

PDF

682

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)。"

[1] Elbein A D, Pan Y T, Irena P, Carroll D . New insights on trehalose: a multifunctional molecule. Glycobiology, 2003,13:17R-27R.
doi: 10.1093/glycob/cwg047 pmid: 12626396
[2] Paul M J, Gonzalez-Uriarte A, Griffiths C A, Hassani-Pak K . The role of trehalose 6-phosphate in crop yield and resilience. Plant Physiol, 2018,177:12-23.
doi: 10.1104/pp.17.01634 pmid: 29592862
[3] 陈素丽, 彭瑜, 周华, 于波, 董彦君, 滕胜 . 植物海藻糖代谢及海藻糖-6-磷酸信号研究进展. 植物生理学报, 2014,50:233-242.
Chen S L, Peng Y, Zhou H, Yu B, Dong Y J, Teng S . Research advances in trehalose metabolism and trehalose-6-phosphate signaling in plants. Plant Physiol J, 2014,50:233-242 (in Chinese with English abstract).
[4] 刘姣, 姜大刚 . 作物海藻糖合成相关基因的研究进展. 基因组学与应用生物学, 2014,33:432-437.
Liu J, Jiang D G . Research advance on trehalose synthesis related genes in crops. Genomics Appl Biol, 2014,33:432-437 (in Chinese with English abstract).
[5] 谢冬微, 王晓楠, 付连双, 孙健, 李卓夫, 郑伟 . 外源海藻糖对冬小麦低温下胚芽长及幼苗抗寒性的影响. 麦类作物学报, 2015,35:215-223.
Xie D W, Wang X N, Fu L S, Sun J, Li Z F, Zheng W . Effect of exogenous trehalose on germ length and seedling freeze resistance of winter wheat under cold stress. J Triticeae Crops, 2015,35:215-223 (in Chinese with English abstract).
[6] 刘旋, 田礼欣, 佟昊阳, 左师宇, 李晶 . 低温胁迫下玉米幼苗根系受外源海藻糖调控的生理表现. 生态学杂志, 2018,37:2354-2361.
Liu X, Tian L X, Tong H Y, Zuo S Y, Li J . Physiological manifestations of maize seedling roots regulated by exogenous trehalose under low temperature stress. Chin J Ecol, 2018,37:2354-2361 (in Chinese with English abstract).
[7] 田礼欣, 曲丹阳, 毕文双, 谢腾龙, 李晶 . 海藻糖对盐胁迫下玉米幼苗生长及生理特性的影响. 草业学报, 2017,26(8):131-138.
Tian L X, Qu D Y, Bi W S, Xie T L, Li J . Trehalose alleviates the negative effects of salinity on the growth and physiological characteristics of maize seedlings. Acta Pratacult Sin, 2017,26(8):131-138 (in Chinese with English abstract).
[8] 巩涛, 刘德海, 王继雯, 杨文玲, 解复红 . 海藻糖合成途径及分子生物学研究进展. 中国农学通报, 2016,32(14):62-67.
Gong T, Liu D H, Wang J W, Yang W L, Xie F H . Advances in trehalose biosynthesis pathways and application of molecular biology technique. Chin Agric Sci Bull, 2016,32(14):62-67 (in Chinese with English abstract).
[9] Vandesteene L, Dijck P V . Expansive evolution of the trehalose-6-phosphate phosphatase gene family in Arabidopsis. Plant Physiol, 2012,160:884.
doi: 10.1104/pp.112.201400 pmid: 22855938
[10] Vogel G, Aeschbacher R A, Müller J, Boller T, Wiemken A . Trehalose-6-phosphate phosphatases from Arabidopsis thaliana: identification by functional complementation of the yeast tps2 mutant. Plant J, 1998,13:673-683.
doi: 10.1046/j.1365-313x.1998.00064.x pmid: 9681009
[11] Krasensky J, Broyart C, Rabanal F A, Jonak C . The redox-sensitive chloroplast trehalose-6-phosphate phosphatase AtTPPD regulates salt stress tolerance. Antiox Redox Signal, 2011,21:1289-1304.
doi: 10.1089/ars.2013.5693
[12] Pramanik M H R, Imai R . Functional identification of a trehalose-6-phosphate phosphatase gene that is involved in transient induction of trehalose biosynthesis during chilling stress in rice. Plant Mol Biol, 2005,58:751-762.
doi: 10.1007/s11103-005-7404-4
[13] Ge L F, Chao D Y, Shi M, Zhu M Z, Gao J P, Lin H X . Overexpression of the trehalose-6-phosphate phosphatase gene OsTPP1 confers stress tolerance in rice and results in the activation of stress responsive genes. Planta, 2008,228:191-201.
doi: 10.1007/s00425-008-0729-x
[14] Satoshi I, Masatomo K, Teruaki T, Masaaki N, Motoaki S, Tomohiko K, Satoshi T, Yoshitaka K, Kazuko Yamaguchi S, Kazuo S . Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenodi dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J, 2001,27:325-333.
doi: 10.1046/j.1365-313x.2001.01096.x pmid: 11532178
[15] 魏开发, 贾文锁, 林子英, 蔡月琴 . NCED3基因雌二醇诱导表达对ABA合成酶基因和代谢酶基因表达的影响. 湖北民族学院学报(自然科学版), 2009,27(1):70-75.
Wei K F, Jia W S, Lin Z Y, Cai Y Q . Effect of NCED3 gene estradiol-induced expression on ABA biosynthetic genes and metabolic enzyme gene expression. J Hubei Univ Nat (Nat Sci Edn), 2009,27(1):70-75 (in Chinese with English abstract).
[16] Hwang S G, Chen H C, Huang W Y, Chu Y C, Shii C T, Cheng W H . Ectopic expression of rice OSNCED3 in Arabidopsis increase ABA level and alters leaf morphology. Plant Sci, 2010,178:12-22.
doi: 10.1016/j.plantsci.2009.09.014
[17] 李辉, 李德芳, 陈安国, 唐慧娟, 李建军, 黄思齐 . 红麻液泡膜质子泵H+-PPase (Hcvp1)基因的克隆、序列分析和表达. 华北农学报, 2017,32(1):9-14.
Li H, Li D F, Chen A G, Tang H J, Li J J, Huang S Q . Cloning and analysis of sequence and expression of a vaculoar H+-PPase gene in Kenaf . Acta Agric Boreali-Sin, 2017,32(1):9-14 (in Chinese with English abstract).
[18] 李辉, 李德芳, 陈安国, 唐慧娟, 李建军, 黄思齐 . 盐和干旱胁迫下红麻HcWD40-1基因的克隆及表达分析. 农业生物技术学报, 2017,25:1970-1978.
Li H, Li D F, Chen A G, Tang H J, Li J J, Huang S Q . Cloning and expression characteristics of HcWD40-1 gene under salt and drought stress in kenaf (Hibiscus cannabinus). J Agric Biotechnol, 2017,25:1970-1978 (in Chinese with English abstract).
[19] Li H, Li D F, Chen A G, Tang H J, Li J J, Huang S Q . RNA-seq for comparative transcript profiling of kenaf under salinity stress. J Plant Res, 2017,130:365-372.
doi: 10.1007/s10265-016-0898-9 pmid: 27999968
[20] 李昉峻, 周其文, 漆新华, 宋正国 . 海藻糖对镉胁迫下水稻幼苗生长的影响. 农业环境科学学报, 2019,38:1827-1834.
Li F J, Zhou Q W, Qi X H, Song Z G . Effects of trehalose on the growth of rice seedlings under cadmium stress. J Agron-Environ Sci, 2019,38:1827-1834 (in Chinese with English abstract).
[21] 庞椿朋, 叶亮, 马健, 路涛, 杨宗艺, 齐明芳 . 海藻糖对高温下番茄幼苗叶片光合作用的调控作用. 江苏农业科学, 2017,45(21):143-146.
Pang C P, Ye L, Ma J, Lu T, Yang Z Y, Qi M F . Regulation of trehalose on photosynthesis of tomato seedling leaves under high temperature. Jiangsu Agric Sci, 2017,45(21):143-146 (in Chinese with English abstract).
[22] 杨雪, 崔喜艳, 李艳丽, 杨美英 . 水稻OsTPP3基因的克隆及生物信息学分析. 分子植物育种, 2018,16:3785-3793.
Yang X, Cui X Y, Li Y L, Yang M Y . Cloning and bioinformatics analysis of OsTPP3 gene in Oryza sativa. Mol Plant Breed, 2018,16:3785-3793 (in Chinese with English abstract).
[23] 丁泽红, 吴春来, 颜彦, 付莉莉, 胡伟 . 木薯海藻糖-6-磷酸酯酶MeTPP6基因克隆及其表达分析, 江苏农业科学, 2019,47(6):31-35.
Ding Z H, Wu C L, Yan Y, Fu L L, Hu W . Cloning and expression analysis of trehalose-6-phosphatase MeTPP6 gene from cassava. Jiangsu Agric Sci, 2019,47(6):31-35 (in Chinese with English abstract).
[24] 刘辉, 王涛涛, 张俊红, 欧阳波, 李汉霞 . 番茄低温响应转录因子SlNAC41克隆及表达分析. 植物生理学报, 2014,50:1707-1716.
Liu H, Wang T T, Zhang J H, Ou-Yang B, Li H X . Cloning and expression analysis of a cold-responsive transcription factor SlNAC41 in tomato. Plant Physiol J, 2014,50:1707-1716 (in Chinese with English abstract).
[1] 颜佳倩, 顾逸彪, 薛张逸, 周天阳, 葛芊芊, 张耗, 刘立军, 王志琴, 顾骏飞, 杨建昌, 周振玲, 徐大勇. 耐盐性不同水稻品种对盐胁迫的响应差异及其机制[J]. 作物学报, 2022, 48(6): 1463-1475.
[2] 雷新慧, 万晨茜, 陶金才, 冷佳俊, 吴怡欣, 王家乐, 王鹏科, 杨清华, 冯佰利, 高金锋. 褪黑素与2,4-表油菜素内酯浸种对盐胁迫下荞麦发芽与幼苗生长的促进效应[J]. 作物学报, 2022, 48(5): 1210-1221.
[3] 戴良香, 徐扬, 张冠初, 史晓龙, 秦斐斐, 丁红, 张智猛. 花生根际土壤细菌群落多样性对盐胁迫的响应[J]. 作物学报, 2021, 47(8): 1581-1592.
[4] 李增强, 丁鑫超, 卢海, 胡亚丽, 岳娇, 黄震, 莫良玉, 陈立, 陈涛, 陈鹏. 铅胁迫下红麻生理特性及DNA甲基化分析[J]. 作物学报, 2021, 47(6): 1031-1042.
[5] 周步进, 李刚, 金刚, 周瑞阳, 刘冬梅, 汤丹峰, 廖小芳, 刘一丁, 赵艳红, 王颐宁. 利用红麻HcPDIL5-2a非全长基因创制雄性不育新种质[J]. 作物学报, 2021, 47(6): 1043-1053.
[6] 李辉, 李德芳, 邓勇, 潘根, 陈安国, 赵立宁, 唐慧娟. 红麻非生物逆境胁迫响应基因HCWRKY71表达分析及转化拟南芥[J]. 作物学报, 2021, 47(6): 1090-1099.
[7] 卢海, 李增强, 唐美琼, 罗登杰, 曹珊, 岳娇, 胡亚丽, 黄震, 陈涛, 陈鹏. 红麻DNA甲基化响应镉胁迫及甲基化差异基因的表达分析[J]. 作物学报, 2021, 47(12): 2324-2334.
[8] 刘亚文, 张红燕, 曹丹, 李兰芝. 基于多平台基因表达数据的水稻干旱和盐胁迫相关基因预测[J]. 作物学报, 2021, 47(12): 2423-2439.
[9] 韦还和, 张徐彬, 葛佳琳, 陈熙, 孟天瑶, 杨洋, 熊飞, 陈英龙, 戴其根. 盐胁迫对水稻颖花形成及籽粒充实的影响[J]. 作物学报, 2021, 47(12): 2471-2480.
[10] 辛正琦, 代欢欢, 辛余凤, 何潇, 谢海艳, 吴能表. 盐胁迫下外源2,4-表油菜素内酯对颠茄氮代谢及TAs代谢的影响[J]. 作物学报, 2021, 47(10): 2001-2011.
[11] 韦还和,葛佳琳,张徐彬,孟天瑶,陆钰,李心月,陶源,丁恩浩,陈英龙,戴其根. 盐胁迫下粳稻品种南粳9108分蘖特性及其与群体生产力的关系[J]. 作物学报, 2020, 46(8): 1238-1247.
[12] 李润枝, 靳晴, 李召虎, 王晔, 彭真, 段留生. 水杨酸提高甘草种子萌发和幼苗生长对盐胁迫耐性的效应[J]. 作物学报, 2020, 46(11): 1810-1816.
[13] 陈晓晶,刘景辉,杨彦明,赵洲,徐忠山,海霞,韩宇婷. 盐胁迫对燕麦叶片生理指标和差异蛋白组学的影响[J]. 作物学报, 2019, 45(9): 1431-1439.
[14] 李旭凯,李任建,张宝俊. 利用WGCNA鉴定非生物胁迫相关基因共表达网络[J]. 作物学报, 2019, 45(9): 1349-1364.
[15] 宋松泉,刘军,徐恒恒,张琪,黄荟,伍贤进. 乙烯的生物合成与信号及其对种子萌发和休眠的调控[J]. 作物学报, 2019, 45(7): 969-981.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2