欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2020, Vol. 46 ›› Issue (10): 1474-1484.doi: 10.3724/SP.J.1006.2020.04014

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

Bna-novel-miR311-HSC70-1模块调控甘蓝型油菜响应热胁迫的机制

鲁海琴1(), 陈丽1,2, 陈磊1, 张盈川1, 文静1, 易斌1, 涂金星1, 傅廷栋1, 沈金雄1,*()   

  1. 1 华中农业大学作物遗传改良国家重点实验室 / 国家油菜工程技术研究中心, 湖北武汉 430070
    2 长江师范学院现代农业与生物工程学院, 重庆 408100
  • 收稿日期:2020-01-16 接受日期:2020-06-02 出版日期:2020-10-12 网络出版日期:2020-06-22
  • 通讯作者: 沈金雄
  • 作者简介:E-mail: 857929189@qq.com
  • 基金资助:
    国家自然科学基金项目(31571698)

Mechanism research of Bna-novel-miR311-HSC70-1 module regulating heat stress response in Brassica napus L.

LU Hai-Qin1(), CHEN Li1,2, CHEN Lei1, ZHANG Ying-Chuan1, WEN Jing1, YI Bin1, TU Jing-Xing1, FU Ting-Dong1, SHEN Jin-Xiong1,*()   

  1. 1 National Key Laboratory of Crop Genetic Improvement / National Engineering Research Center of Rapeseed, Huazhong Agriculatural University, Wuhan 430070, Hubei, China
    2 School of Advanced Agriculture and Bioengineering, Yangtze Normal University, Chongqing 408100, China
  • Received:2020-01-16 Accepted:2020-06-02 Published:2020-10-12 Published online:2020-06-22
  • Contact: Jin-Xiong SHEN
  • Supported by:
    National Natural Science Foundation of China(31571698)

RichHTML

28

PDF

572

摘要：

HSP70 (heat shock protein 70)参与植物热胁迫应答, 增强植物耐热性, 但目前油菜中尚无miRNA调控HSP70基因的报道。本研究novel-miR311是利用高通量技术在甘蓝型油菜茎尖中筛选出的新miRNA。novel-miR311存在于油菜而不存在于拟南芥中, 5°-RACE技术证实其2个靶基因属热应激同源蛋白基因HSC70-1 (HSP70家族), 在甘蓝型油菜体内被剪切。构建novel-miR311超表达载体, 转化拟南芥和甘蓝型油菜, 其转基因阳性苗中HSC70-1基因表达量显著下降。高温胁迫试验表明, 拟南芥和甘蓝型油菜热胁迫后, 其阳性苗的生长势和存活率显著低于其对应的对照。qPCR结果显示, 油菜中HSC70-1基因表达量热胁迫后较热胁迫前上升。上述结果表明, 油菜novel-miR311介导HSC70-1基因的剪切降低了拟南芥和甘蓝型油菜耐热性。

关键词: 甘蓝型油菜, 拟南芥, novel-miR311, HSC70-1, 高温胁迫

Abstract:

HSP70 (heat shock protein 70) participates in the response to heat stress, and can enhance plant heat tolerance, but there have been no reports of miRNA regulating HSP70 in rapeseed. In this study, a new miRNA, named novel-miR311, was identified in the shoot tip of Brassica napus by high-throughput technology. novel-miR311 was present in Brassica napus but not in Arabidopsis, and 5°-RACE technology confirmed that its two target genes, belonged to heat stress homologous protein gene HSC70-1 (HSP70 family), and could be cleavaged in Brassica napus. An overexpression vector of novel-miR311 was constructed and transformed into Arabidopsis and Brassica napus, and the expression of HSC70-1 in transgenic positive seedlings was decreased significantly. High temperature stress experiments showed that the growth potential and survival rates of Arabidopsis and Brassica napus positive seedlings were lower than those of their corresponding controls. The qPCR results showed that the expression of HSC70-1 gene in rapeseed increased after heat stress than before stress. In conclusion, the results suggest that Bna- novel-miR311 could reduce the heat resistance of Arabidopsis and Brassica napus by mediating cleavaged of HSC70-1.

Key words: Brassica napus, Arabidopsis thaliana, novel-miR311, HSC70-1, high temperature stress

表1

novel-miR311降解组测序预测到的10个候选靶基因"

miRNA 名称
miRNA name		 靶基因
Target gene		 拟南芥同源基因
Arabidopsis homologous gene		 靶基因功能
Function of target gene
novel miR311 BnaC06g18840D AT3G63460.1 Transducin family protein
novel miR311 BnaA07g19590D AT3G63460.1 Transducin family protein
novel miR311 BnaC03g46700D AT5G02490.1 Heat shock protein 70 (Hsp 70) family protein
novel miR311 BnaA03g39360D AT5G02490.1 Heat shock protein 70 (Hsp 70) family protein
novel miR311 BnaA03g39350D AT5G02490.1 Heat shock protein 70 (Hsp 70) family protein
novel miR311 BnaC04g42010D AT5G02500.1 Heat shock cognate protein 70-1 (HSC70-1)
novel miR311 BnaA10g27080D AT5G02500.1 Heat shock cognate protein 70-1 (HSC70-1)
novel miR311 BnaA09g05850D AT5G02500.1 Heat shock cognate protein 70-1 (HSC70-1)
novel miR311 BnaCnng03470D AT5G02500.1 Heat shock cognate protein 70-1 (HSC70-1)
novel miR311 BnaA10g27060D AT5G02500.1 Heat shock cognate protein 70-1 (HSC70-1)

图1

5°-RACE验证靶基因 冒号表示碱基配对, 点号表示错配, 箭头表示切割位点, 数字表示挑斑克隆。"

图2

油菜和拟南芥靶基因作用位点序列对比 a: 转导蛋白基因; b: 热激蛋白HSP70-2基因; c: 热应激同源蛋白HSC70-1基因。红色部分为novel-miR311与靶基因结合部位。"

图3

野生型和阳性拟南芥中novel-miR311和HSC70-1分别在根、茎、叶、花和角果中的表达量 每次试验3次生物学重复。*和**分别表示P < 0.05和P < 0.01。"

图4

野生型和阳性拟南芥在1/2MS上的高温胁迫实验 a: 表型分析; b: 统计学分析。"

图5

甘蓝型油菜阳性苗与J572的种子高温胁迫后的发芽试验 a: 不同温度处理前后对比图; b: 2 d发芽率统计图。"

图6

甘蓝型油菜阳性苗与J572在土壤中的高温胁迫表型及表达分析 a: 热胁迫前后阳性苗与J572的对比图, 图中红色箭头指存活的油菜。b: 热胁迫前后novel-miR311和HSC70-1的表达量。每次试验3次生物学重复。**表示P < 0.01。"

图7

HSP70作用途径 根据Jacob等[33]并略作修改, 虚框中为引入本文结果。"

附图1

novel-miR311茎环结构"

附表1

novel-miR311的前体和成熟体序列"

类别Type 序列Sequence (5°-3°)
成熟体
Mature sequence (5′-3′)		 TTGGTGATAATTGGATTGGCA
前体
Precursor sequence (5′-3′)		 TATGTGGTTAGAGCCAATCCATTTATCACCAATTCGTTTAAGTTGGTATAAATGTCGGTTTAAGAGCGATCAGGCCTATCAAAAATAGGCCGGATCGAATTCTGTTCAGTTGGGCAAGGTGTGTTTGTGCTCTGATACCATGATAAATTTCTTAGTTTTACAATTAAAACTAATTGGTGATAATTGGATTGGCACGTCCCTAAC
引物
Primer (5′-3′)		 5' primer: AACTGCAGTTGTAGTTTTGAGAGATTAGAAGTGG
3' primer: CGGGATCCCTTTTATTAATCCCTCAGTAATACACC
[1] Sung D Y, Vierling E, Guy C L. Comprehensive expression profile analysis of the Arabidopsis Hsp70 gene family. Plant Physiol, 2001,126:789-800.
doi: 10.1104/pp.126.2.789 pmid: 11402207
[2] Usman M G, Rafii M Y, Martini M Y, Yusuff O A, Ismail M R, Miah G. Molecular analysis of Hsp70 mechanisms in plants and their function in response to stress. Biotechnol Genet Eng Rev, 2017,33:26-39.
doi: 10.1080/02648725.2017.1340546 pmid: 28649918
[3] Flaherty K M, Wilbanks S M, DeLuca Flaherty C, McKay D B. Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. II. Structure of the active site with ADP or ATP bound to wild type and mutant ATPase fragment. J Biol Chem, 1994,269:12899-12907.
pmid: 8175707
[4] Meimaridou E, Gooljar S B, Chapple J P. From hatching to dispatching: the multiple cellular roles of the Hsp70 molecular chaperone machinery. J Mol Endocrinol, 2008,42:1-9.
doi: 10.1677/JME-08-0116 pmid: 18852216
[5] 孔凡英, 邓永胜, 孟庆伟. 叶绿体J蛋白研究进展. 植物生理学报, 2011,47:235-243.
Kong F Y, Deng Y S, Meng Q W. Research progress of chloroplast J protein. Plant Physiol J, 2011,47:235-243 (in Chinese with English abstract).
[6] Murphy M E. The HSP70 family and cancer. Carcinogenesis, 2013,34:1181-1188.
doi: 10.1093/carcin/bgt111
[7] 陈丽, 鲁海琴, 李日慧, 傅廷栋, 沈金雄. 油菜miRNA研究现状与展望. 中国油料作物学报, 2018,40:664-673.
Chen L, Lu H Q, Li R H, Fu T D, Shen J X. Research progress and prospect of miRNA in Brassica napus. Chin J Oil Crop Sci, 2018,40:664-673 (in Chinese with English abstract).
[8] Reinhart B J, Weinstein E G, Rhoades M W, Bartel B, Bartel D P. MicroRNAs in plants. Genes Dev, 2002,16:1616-1626
doi: 10.1101/gad.1004402 pmid: 12101121
[9] Huang J, Li Z, Zhao D. Deregulation of the OsmiR160 target gene OsARF18 causes growth and developmental defects with an alteration of auxin signaling in rice. Sci Rep, 2016,6:29938.
doi: 10.1038/srep29938 pmid: 27444058
[10] Waters B M, McInturf S A, Stein R J. Rosette iron deficiency transcript and microRNA profiling reveals links between copper and iron homeostasis in Arabidopsis thaliana. J Exp Bot, 2012,63:5903-5918.
doi: 10.1093/jxb/ers239
[11] Zhang H, Zhao X, Li J, Cai H, Deng X W, Li L. MicroRNA408 is critical for the HY5-SPL7 gene network that mediates the coordinated response to light and copper. Plant Cell, 2014,26:4933-4953.
doi: 10.1105/tpc.114.127340
[12] Srivastava S, Srivastava A K, Suprasanna P, D’Souza S F. Identification and profiling of arsenic stress-induced microRNAs in Brassica juncea. J Exp Bot, 2013,64:303-315.
doi: 10.1093/jxb/ers333 pmid: 23162117
[13] Meng J G, Zhang X D, Tan S K, Zhao K X, Yang Z M. Genome-wide identification of Cd-responsive NRAMP transporter genes and analyzing expression of NRAMP1 mediated by miR167 in Brassica napus. Biometals, 2017,30:1-15.
doi: 10.1007/s10534-016-9981-x pmid: 27853903
[14] Sunkar R, Kapoor A, Zhu J K. Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance. Plant Cell, 2006,18:2415-2415.
doi: 10.1105/tpc.106.180960
[15] Chen L, Chen L, Zhang X, Liu T, Niu S, Wen J, Yi B, Ma C, Tu J, Fu T, Shen J. Identification of miRNAs that regulate silique development in Brassica napus. Plant Sci, 2018,269:106-117.
doi: 10.1016/j.plantsci.2018.01.010 pmid: 29606207
[16] Kumar R R, Pathak H, Sharma S K, Kala Y K, Nirjal M K, Singh G P, Goswami S, Rai R D. Novel and conserved heat-responsive microRNAs in wheat (Triticum aestivum L.). Funct Integr Genomics, 2015,15:323-348.
doi: 10.1007/s10142-014-0421-0 pmid: 25480755
[17] Pan C, Ye L, Zheng Y, Wang Y, Yang D, Liu X, Chen L, Zhang Y, Fei Z, Lu G. Identification and expression profiling of microRNAs involved in the stigma exsertion under high- temperature stress in tomato. BMC Genomics, 2017,18:843.
doi: 10.1186/s12864-017-4238-9 pmid: 29096602
[18] Zhang M, An P, Li H, Wang X, Zhou J, Dong P, Zhao Y, Wang Q, Li C. The miRNA-mediated post-transcriptional regulation of maize in response to high temperature. Int J Mol Sci, 2019,20:1754.
doi: 10.3390/ijms20071754
[19] Zhou R, Wang Q, Jiang F, Cao X, Sun M, Liu M, Wu Z. Identification of miRNAs and their targets in wild tomato at moderately and acutely elevated temperatures by high-throughput sequencing and degradome analysis. Sci Rep, 2016,6:33777.
doi: 10.1038/srep33777 pmid: 27653374
[20] Shi X, Jiang F, Wen J, Wu Z. Overexpression of solanum habrochaites microRNA319d (sha-miR319d) confers chilling and heat stress tolerance in tomato (S. lycopersicum). BMC Plant Biol, 2019,19:214.
pmid: 31122194
[21] Ding Y, Ma Y, Liu N, Xu J, Hu Q, Li Y, Wu Y, Xie S, Zhu L, Min L, Zhang X. MicroRNAs involved in auxin signalling modulate male sterility under high-temperature stress in cotton (Gossypium hirsutum). Plant J, 2017,91:977-994.
doi: 10.1111/tpj.13620 pmid: 28635129
[22] Stief A, Altmann S, Hoffmann K, Pant B D, Scheible W R, Bäurle I. Arabidopsis miR156 regulates tolerance to recurring environmental stress through SPL transcription factors. Plant Cell, 2014,26:1792-1807.
doi: 10.1105/tpc.114.123851
[23] Matthews C, Arshad M, Hannoufa A. Alfalfa response to heat stress is modulated by microRNA156. Physiol Plant, 2019,165:830-842
doi: 10.1111/ppl.12787 pmid: 29923601
[24] 焦聪聪, 黄吉祥, 汪义龙, 张晓玉, 熊化鑫, 倪西源, 赵坚义. 利用非条件和条件QTL解析油菜产量相关性状的遗传关系. 作物学报, 2015,41:1481-1489.
doi: 10.3724/SP.J.1006.2015.01481
Jiao C C, Huang J X, Wang Y L, Zhang X Y, Xiong H X, Ni X Y, Zhao J Y. Genetic analysis of yield-associated traits by unconditional and conditional QTL in Brassica napus. Acta Agron Sin, 2015,41:1481-1489 (in Chinese with English abstract).
[25] Staff T P O. Correction: a genome-wide perspective of miRNAome in response to high temperature, salinity and drought stresses in Brassica juncea (Czern) L. PLoS One, 2015,10:e92456.
[26] Yu X, Wang H, Lu Y, de Ruiter M, Cariaso M, Prins M, van Tunen A, He Y. Identification of conserved and novel microRNAs that are responsive to heat stress in Brassica rapa. J Exp Bot, 2012,63:1025-1038.
doi: 10.1093/jxb/err337
[27] 陈丽. 甘蓝型油菜株型及角果长度相关miRNA和靶基因的挖掘. 华中农业大学博士学位论文, 湖北武汉, 2018.
Chen L. The Study of miRNA and Targets Regulate Plant Architecture and Silique Length in Brassica napus L. PhD Dissertation of Huazhong Agricultural University, Wuhan, Hubei, China, 2018 (in Chinese with English abstract).
[28] Varkonyi Gasic E, Wu R, Wood M, Walton E F, Hellens R P. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods, 2007,3:12.
doi: 10.1186/1746-4811-3-12 pmid: 17931426
[29] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method . Methods, 2001,25:402-408.
doi: 10.1006/meth.2001.1262 pmid: 11846609
[30] Leng L, Liang Q, Jiang J, Zhang C, Hao Y, Wang X, Su W. A subclass of HSP70s regulate development and abiotic stress responses in Arabidopsis thaliana. J Plant Res, 2017,130:349-363.
doi: 10.1007/s10265-016-0900-6 pmid: 28004282
[31] Wang W, Vinocur B, Shoseyov O, Altman A. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci, 2004,9:244-252.
doi: 10.1016/j.tplants.2004.03.006 pmid: 15130550
[32] 胡秀丽, 李艳辉, 杨海荣, 刘全军, 李潮海. HSP70可提高干旱高温复合胁迫诱导的玉米叶片抗氧化防护能力. 作物学报, 2010,36:636-644.
doi: 10.3724/SP.J.1006.2010.00636
Hu X L, Li Y H, Yang H R, Liu Q J, Li C H. Heat shock protein 70 may improve the ability of antioxidant defense induced by the combination of drought and heat in maize leaves. Acta Agron Sin, 2010,36:636-644 (in Chinese with English abstract).
[33] Jacob P, Hirt H, Bendahmane A. The heat shock protein/chaperone network and multiple stress resistance. Plant Biotechnol J, 2017,15:405-414.
doi: 10.1111/pbi.12659 pmid: 27860233
[34] Scharf K D, Berberich T, Ebersberger I, Nover L. The plant heat stress transcription factor (Hsf) family: structure, function and evolution. Biochim Biophys Acta, 2012,1819:104-119.
doi: 10.1016/j.bbagrm.2011.10.002 pmid: 22033015
[35] Sung D Y, Guy C L. Physiological and molecular assessment of altered expression of Hsc70-1 in Arabidopsis. evidence for pleiotropic consequences. Plant Physiol, 2003,132:979-987.
doi: 10.1104/pp.102.019398 pmid: 12805626
[36] Cazalé A C, Clément M, Chiarenza S, Roncato M A, Pochon N, Creff A, Marin E, Leonhardt N, Noël L D. Altered expression of cytosolic/nuclear HSC70-1 molecular chaperone affects development and abiotic stress tolerance in Arabidopsis thaliana. J Exp Bot, 2009,60:2653-2664.
doi: 10.1093/jxb/erp109 pmid: 19443614
[37] Young L W, Wilen R W, Bonham Smith P C. High temperature stress of Brassica napus during flowering reduces micro- and megagametophyte fertility, induces fruit abortion, and disrupts seed production. J Exp Bot, 2004,55:485-495.
doi: 10.1093/jxb/erh038 pmid: 14739270
[38] Wang X, Yan B, Shi M, Zhou W, Zekria D, Wang H, Kai G. Overexpression of a Brassica campestris HSP70 in tobacco confers enhanced tolerance to heat stress. Protoplasma, 2016,253:637-645.
doi: 10.1007/s00709-015-0867-5 pmid: 26298102
[1] 陈松余, 丁一娟, 孙峻溟, 黄登文, 杨楠, 代雨涵, 万华方, 钱伟. 甘蓝型油菜BnCNGC基因家族鉴定及其在核盘菌侵染和PEG处理下的表达特性分析[J]. 作物学报, 2022, 48(6): 1357-1371.
[2] 秦璐, 韩配配, 常海滨, 顾炽明, 黄威, 李银水, 廖祥生, 谢立华, 廖星. 甘蓝型油菜耐低氮种质筛选及绿肥应用潜力评价[J]. 作物学报, 2022, 48(6): 1488-1501.
[3] 袁大双, 邓琬玉, 王珍, 彭茜, 张晓莉, 姚梦楠, 缪文杰, 朱冬鸣, 李加纳, 梁颖. 甘蓝型油菜BnMAPK2基因的克隆及功能分析[J]. 作物学报, 2022, 48(4): 840-850.
[4] 黄成, 梁晓梅, 戴成, 文静, 易斌, 涂金星, 沈金雄, 傅廷栋, 马朝芝. 甘蓝型油菜BnAPs基因家族成员全基因组鉴定及分析[J]. 作物学报, 2022, 48(3): 597-607.
[5] 王瑞, 陈雪, 郭青青, 周蓉, 陈蕾, 李加纳. 甘蓝型油菜白花基因InDel连锁标记开发[J]. 作物学报, 2022, 48(3): 759-769.
[6] 孟颖, 邢蕾蕾, 曹晓红, 郭光艳, 柴建芳, 秘彩莉. 小麦Ta4CL1基因的克隆及其在促进转基因拟南芥生长和木质素沉积中的功能[J]. 作物学报, 2022, 48(1): 63-75.
[7] 赵文青, 徐文正, 杨锍琰, 刘玉, 周治国, 王友华. 棉花叶片响应高温的差异与夜间淀粉降解密切相关[J]. 作物学报, 2021, 47(9): 1680-1689.
[8] 王艳花, 刘景森, 李加纳. 整合GWAS和WGCNA筛选鉴定甘蓝型油菜生物产量候选基因[J]. 作物学报, 2021, 47(8): 1491-1510.
[9] 李杰华, 端群, 史明涛, 吴潞梅, 柳寒, 林拥军, 吴高兵, 范楚川, 周永明. 新型抗广谱性除草剂草甘膦转基因油菜的创制及其鉴定[J]. 作物学报, 2021, 47(5): 789-798.
[10] 唐鑫, 李圆圆, 陆俊杏, 张涛. 甘蓝型油菜温敏细胞核雄性不育系160S花药败育的形态学特征和细胞学研究[J]. 作物学报, 2021, 47(5): 983-990.
[11] 周新桐, 郭青青, 陈雪, 李加纳, 王瑞. GBS高密度遗传连锁图谱定位甘蓝型油菜粉色花性状[J]. 作物学报, 2021, 47(4): 587-598.
[12] 李书宇, 黄杨, 熊洁, 丁戈, 陈伦林, 宋来强. 甘蓝型油菜早熟性状QTL定位及候选基因筛选[J]. 作物学报, 2021, 47(4): 626-637.
[13] 张春, 赵小珍, 庞承珂, 彭门路, 王晓东, 陈锋, 张维, 陈松, 彭琦, 易斌, 孙程明, 张洁夫, 傅廷栋. 甘蓝型油菜千粒重全基因组关联分析[J]. 作物学报, 2021, 47(4): 650-659.
[14] 唐婧泉, 王南, 高界, 刘婷婷, 文静, 易斌, 涂金星, 傅廷栋, 沈金雄. 甘蓝型油菜SnRK基因家族生物信息学分析及其与种子含油量的关系[J]. 作物学报, 2021, 47(3): 416-426.
[15] 蒙姜宇, 梁光伟, 贺亚军, 钱伟. 甘蓝型油菜耐盐和耐旱相关性状的QTL分析[J]. 作物学报, 2021, 47(3): 462-471.
Viewed
Full text


Abstract

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