欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2021, Vol. 47 ›› Issue (8): 1417-1426.doi: 10.3724/SP.J.1006.2021.01071

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

小麦lncRNA27195及其靶基因TaRTS克隆及表达分析

王娜1,2(), 白建芳2, 马有志1, 郭昊宇2, 王永波2, 陈兆波2, 赵昌平2,*(), 张立平2,*()   

  1. 1中国农业科学院作物科学研究所, 北京 100087
    2北京市农林科学院北京杂交小麦工程技术研究中心/杂交小麦分子遗传北京市重点实验室, 北京 100097
  • 收稿日期:2020-09-02 接受日期:2021-01-13 出版日期:2021-08-12 网络出版日期:2021-02-22
  • 通讯作者: 赵昌平,张立平
  • 作者简介:王娜, E-mail: wangna_00@sina.com
  • 基金资助:
    北京市自然科学基金项目(6182014);北京市农林科学院杰出科学家项目(JKZX201907);北京市农林科学院青年基金(QNJJ201916);国家自然科学基金项目(31872881);北京市农林科学院科技创新能力建设专项(KJCX20180403)

Cloning and expression analysis of lncRNA27195 and its target gene TaRTS in wheat (Triticum aestivum L.)

WANG Na1,2(), BAI Jian-Fang2, MA You-Zhi1, GUO Hao-Yu2, WANG Yong-Bo2, CHEN Zhao-Bo2, ZHAO Chang-Ping2,*(), ZHANG Ling-Ping2,*()   

  1. 1Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100087, China
    2Beijing Engineering and Technique Research Center for Hybrid Wheat, Beijing Academy of Agriculture and Forestry Sciences/Municipal Key Laboratory of the Molecular Genetics of Hybrid Wheat, Beijing 100097, China
  • Received:2020-09-02 Accepted:2021-01-13 Published:2021-08-12 Published online:2021-02-22
  • Contact: ZHAO Chang-Ping,ZHANG Ling-Ping
  • Supported by:
    Natural Science Foundation of Beijing(6182014);Outstanding Scientist Cultivation Program of BAAFS(JKZX201907);Foundation for Youths of BAAFS(QNJJ201916);National Natural Science Foundation of China(31872881);Special Project of Science and Technology Innovation Ability Construction of BAAFS(KJCX20180403)

RichHTML

43

PDF

917

摘要：

长链非编码RNA (long non-coding RNA, lncRNA)是一种大于200 bp的非编码RNA, 大量存在于植物体中, 其可通过调节基因表达或蛋白功能, 在植物生长发育与胁迫应答中发挥重要作用。前期在研究光照和温度对小麦光温敏核雄性不育系BS366育性诱导中, 利用转录组测序筛选获得与育性相关的lncRNA (lncRNA2719)。为研究小麦lncRNA27195的功能, 本研究在BS366中克隆lncRNA27195及其靶基因TaRTS, 并对TaRTS进行生物信息学分析, 结果显示, TaRTS基因全长315 bp, 编码104个氨基酸, 且发现该RTS蛋白仅存在于禾本科植物中。通过实时荧光定量PCR, 对lncRNA27195及TaRTS基因在不同组织不同光温处理及茉莉酸甲酯处理下进行表达模式分析, 发现lncRNA27195和TaRTS均在雄蕊中高表达, 呈显著的正相关, 且两者在不同光温条件下呈现出不同的表达模式。光照和温度均对lncRNA27195和TaRTS有调控作用, 适当浓度的MeJA促进两者的表达, SA抑制两者表达。以上结果表明, 在光周期、温度和植物激素的诱导下, lncRNA27195正向调节TaRTS基因表达, 进而影响花粉育性, 本研究结果有助于促进对小麦光温敏核型雄性不育系的机理研究和生产应用。

关键词: lncRNA, RTS蛋白, 花药, 雄性不育

Abstract:

Long non-coding RNA (lncRNA) is a non-coding RNA length over 200 bp, which is abundant in plants. It plays important roles in plant growth, development, and stress response by regulating gene expression or protein function. In the previous study, a fertility-related lncRNA named lncRNA27195 was screened and obtained by transcriptome sequencing from the anther of wheat Photoperiod-thermo Sensitive Genic Male Sterility (PTGMS) line BS366. To investigate the function of lncRNA27195 in wheat, the lncRNA27195 gene and its target gene TaRTS were cloned from BS366. Bioinformatics analysis were performed on TaRTS. The expressions of lncRNA27195 and TaRTS in different tissues and their expression correlation between them were analyzed by qRT-PCR. Meanwhile, the expression patterns of lncRNA27195 and TaRTS under different light and temperature treatments, and methyl jasmonate (MeJA) treatments were investigated. The results showed that the TaRTS gene with 315 bp length, encoded 104 amino acids. Additionally, RTS proteins were only found as anther-specific proteins in gramineae plants. Both lncRNA27195 and TaRTS with a significantly positive correlation were highly expressed in stamens, and revealed different expression patterns in different fertility environments. The results demonstrated that the expression of lncRNA27195 and TaRTS were also regulated by light and temperature. In addition, we found that the appropriate concentration of MeJA could promote the expression of lncRNA27195 and TaRTS while SA could inhibit the expression. The results indicated that under the induction of photoperiod, temperature, and plant hormones, IncRNA27195 positively regulated TaRTS gene expression, resulting in affecting pollen development and male fertility. This study contributed to the mechanism research and production application of PTGMS wheat.

Key words: lncRNA, RTS protein, anther, male sterility

表1

引物设计"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 产物大小
Product length (bp)
lncRNA27195-F TTTAATCTTCCGAGCTCACCCA 396
lncRNA27195-R TTTAAATGTTAAAACATGTTATATTTTTGA
TaRTS-F TTTAAATGGCACGCACCGGC 315
TaRTS-R TTTAATCAGAGCCCCTGGGTGGT
lncRNA27195-qRT-F GCATACGCACCGCATGTATAAT 109
lncRNA27195-qRT-R CTAGTCGCACAGGTCGACTGAA
TaRTS-qRT-F AGGTCGCATCGTGCCTCCT 114
TaRTS-qRT-R CCCCTGGGTGGTGCAGT
Actin1-F TTGGCATCTCTCAGCACCTTC 138
Actin1-R TCAGCCTTCGCAATCCACA

图1

lncRNA27195基因和TaRTS基因扩增片段(A)和序列(B, C)"

图2

lncRNA27195基因(A)和TaRTS基因(B)序列比对"

图3

TaRTS蛋白的亲水性(A)、二级结构(B)和三级结构(C) 蓝色为α-螺旋, 绿色为β-转角, 红色为延伸链, 紫色为无规则卷曲。"

图4

TaRTS蛋白的小麦原生质体亚细胞定位"

图5

TaRTS的多序列比对(A)和进化树分析(B)"

表2

启动子元件分析"

上游启动子元件 Upstream promoter elements 功能 Function
CAAT-box, TATA-box 核心启动子元件 Core promoter element
ACE, G-box, GT1-motif, T-box, Sp1 光响应元件 Light responsiveness element
ABRE 脱落酸响应元件 Abscisic acid responsiveness element
CAT-box 分生组织表达相关元件 Related to meristem expression element
CCAAT-box MYBHv1结合位点 MYBHv1 binding site
CGTCA-motif, TGACG-motif 茉莉酸甲酯响应元件 MeJA-responsiveness responsiveness element
LTR 低温响应元件 Low-temperature responsiveness element
MSA-like 细胞周期调控元件 Cell cycle regulation element

图6

lncRNA27195基因(A)及TaRTS基因(B)组织特异性表达分析"

图7

TaRTS(A)和lncRNA27195(B)表达模式分析及两者表达量相关性分析(C) *代表显著差异(P<0.05), **代表极显著差异(P<0.01)。"

图8

TaRTS及lncRNA27195在不同光照温度条件下表达模式分析 T1: 温长日环境(20℃, 14 h L/10 h D); T2: 高温短日环境(20℃, 12 h L/12 h D); T3: 低温长日环境(12℃, 14 h L/10 h D); T4为低温短日(12℃, 12 h L/12 h D)环境。*代表各组间存在显著差异(P < 0.05)。"

图9

TaRTS及lncRNA27195在MeJA和SA处理下的表达模式分析"

[1] Espinoza C A, Goodrich J A, Kugel J F. Characterization of the structure, function, and mechanism of B2 RNA, an ncRNA repressor of RNA polymerase II transcription. RNA, 2007,13:583-596.
pmid: 17307818
[2] Sigova A A, Mullen A C, Molinie B, Gupta S, Orlando D A, Guenther M G, Almada A E, Lin C, Sharp P A, Giallourakis C C, Young R A. Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells. Proc Natl Acad Sci USA, 2013,110:2876-2881.
doi: 10.1073/pnas.1221904110
[3] Miao S, Lee K W. From discovery to function: the expanding roles of long non-coding RNAs in physiology and disease. Endocr Rev, 2015,36:25-64.
doi: 10.1210/er.2014-1034
[4] Rinn J L, Kertesz M, Wang J K, Squazzo S L, Xu X, Brugmann S A, Henry Goodnough L, Helms J A, Farnham P J, Segal E, Chang H Y. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell, 2007,129:1311-1323.
doi: 10.1016/j.cell.2007.05.022
[5] Kristina P S, Thomassen M, Tan Q, Martin B, Torben A K. Long non-coding RNA HOTAIR is an independent prognostic marker of metastasis in estrogen receptor-positive primary breast cancer. Breast Cancer Res Treeat, 2013,142:529-536.
[6] Reis E M, Nakaya H I, Louro R, Canavez F C, Flatschart A V, Almeida G T, Egidio C M, Paquola A C, Machado A A, Festa F, Yamamoto D, Alvarenga R, da Silva C C, Brito G C, Simon S D, Moreira Filho C A, Leite K R, Camara Lopes L H, Campos F S, Gimba E, Vignal G M E, Dorry H, Sogayar M C, Barcinski M A, da Silva A M, Verjovski Almeida S. Antisense intronic non- coding RNA levels correlate to the degree of tumor differentiation in prostate cancer. Oncogene, 2004,23:6684-6692.
doi: 10.1038/sj.onc.1207880
[7] Swiezewski S, Liu F, Magusin A, Dean C. Cold-induced silencing by long antisense transcripts of an Arabidopsis Polycomb target. Nature, 2009,462:799-802.
doi: 10.1038/nature08618 pmid: 20010688
[8] Xin M M, Wang Y, Yao Y Y, Song N, Hu Z, Qin D D, Xie C J, Peng H R, Ni Z F, Sun Q X. Identification and characterization of wheat long non-protein coding RNAs responsive to powdery mildew infection and heat stress by using microarray analysis and SBS sequencing. BMC Plant Biol, 2011,11:61.
doi: 10.1186/1471-2229-11-61
[9] Ding J, Lu Q, Ou-Yang Y, Mao H, Zhang P, Yao J, Xu C, Li X, Xiao J, Zhang Q. A long noncoding RNA regulates photoperiod-sensitive male sterility, an essential component of hybrid rice. Proc Natl Acad Sci USA, 2012,109:2654-2659.
doi: 10.1073/pnas.1121374109
[10] Zhou H, Liu Q J, Li J, Jiang D, Zhou L, Wu P, Lu S, Li F, Zhu L, Liu Z, Chen L, Liu Y, Zhuang C. Photoperiod- and thermo- sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA. Cell Res, 2012,22:649-660.
doi: 10.1038/cr.2012.28
[11] Dai X Y, Yu J J, Zhao Q, Zhu D, Ao G. Non-coding RNA for ZM401, a pollen-specific gene of Zea mays. Acta Bot Sin, 2004,46:497-504.
[12] Liu C M, Muchhal U S, Raghothama K G. Differential expression of TPS11, a phosphate starvation-induced gene in tomato. Plant Mol Biol, 1997,33:867-874.
pmid: 9106510
[13] 赵昌平, 王新, 张风廷, 叶志杰, 戴惠君. 杂种小麦的研究现状与光温敏二系法. 北京农业科学, 1999,17(2):3-5.
Zhao C P, Wang X, Zhang F T, Ye Z J, Dai H J. Research status of hybrid wheat and the photosensitive two-system method. Beijing Agric Sci, 1999,17(2):3-5 (in Chinese with English abstract).
[14] Luo H, Lee J Y, Hu Q, Nelson-Vasilchik K, Eitas T K, Lickwar C, Kausch A P, Chandlee J M, Hodges T K. RTS, a rice anther- specific gene is required for male fertility and its promoter sequence directs tissue-specific gene expression in different plant species. Plant Mol Biol, 2006,62:397-408.
doi: 10.1007/s11103-006-9031-0
[15] Kelliher T, Walbot V. Hypoxia triggers meiotic fate acquisition in maize. Science, 2012,337:345-348.
doi: 10.1126/science.1220080
[16] Shi J, Cui M, Yang L, Kim Y J, Zhang D B. Genetic and biochemical mechanisms of pollen wall development. Trends Plant Sci, 2015,20:741-753
doi: 10.1016/j.tplants.2015.07.010
[17] Julian I S. Arabidopsis somatic embryogenesis receptor kinases1 and 2 are essential for tapetum development and microspore maturation. Plant Cell, 2005,17:3350-3361.
doi: 10.1105/tpc.105.036731
[18] Jeon J S, Chung Y Y, Lee S, Yi G H, Oh B G, An G. Isolation and characterization of an anther-specific gene, RA8, from rice (Oryza sativa L.). Plant Mol Biol, 1999,39:35-44.
pmid: 10080707
[19] Xu H, Knox R B, Philip E T, Singh M B. Bcp1, a gene required for male fertility in Arabidopsis. Proc Natl Acad Sci USA, 1995,92:2106-2110.
doi: 10.1073/pnas.92.6.2106
[20] Theerakulpisut P. Isolation and developmental expression of Bcp1, an anther-specific cDNA clone in Brassica campestris. Plant Cell, 1991,3:1073-1084.
pmid: 1821760
[21] Zou J T, Zhan X Y, Wu H M, Wang H, Cheung A Y. Characterization of a rice pollen-specific gene and its expression. Am J Bot, 1994,81:552-561.
doi: 10.1002/j.1537-2197.1994.tb15484.x
[22] Hanson D D, Hamilton A D, Travis L J, Bashe D M, Mascarenhas J P. Characterization of a pollen-specific cDNA clone Zea mays and its expression. Plant Cell, 1989,1:173-179.
pmid: 2535540
[23] Twell D, Wing R, Yamaguchi J, McCormick S. Isolation and expression of an anther-specific gene form tomato. Mol Gen Genet, 1989,217:240-245.
doi: 10.1007/BF02464887
[24] McCormick S. Male gametophyte development. Plant Cell, 1993,5:1265-1275.
doi: 10.2307/3869779
[25] Tsuchiya T, Toriyama K, Nasrallah M E, Ejiri S. Isolation of genes abundantly expressed in rice anthers at the microspore stage. Plant Mol Biol, 1992,20:1189-1193.
pmid: 1463854
[26] Creelman R A, Mullet J E. Biosynthesis and action of jasmonates in plants. Annu Rev Plant Phys, 1997,48:355-381.
[27] Mandaokar A, Thines B, Shin B, Lange B M, Choi G, Koo Y J, Yoo Y J, Choi Y D, Choi G, Browse J. Transcriptional regulators of stamen development in Arabidopsis identified by transcriptional profiling. Plant J, 2006,46:984-1008.
pmid: 16805732
[28] Muruáis G, Lalioti V, Sandoval I V. The Cdk5 inhibitor roscovitine strongly inhibits glucose uptake in 3T3-L1 adipocytes without altering GLUT4 translocation from internal pools to the cell surface. J Cell Physiol, 2009,220:238-244.
doi: 10.1002/jcp.v220:1
[29] Seo S, Okamoto N, Seto H, Ishizuka K, Sano H, Ohashi Y. Tobacco map kinase: a possible mediator in wound signal-transduction pathways. Science, 1995,270:1988-1992.
doi: 10.1126/science.270.5244.1988
[30] Seo S, Sano H, Ohashi Y. Jasmonate-based wound signal transduction requires activation of WIPK, a tobacco mitogen-activated protein kinase. Plant Cell, 1999,11:289-298.
pmid: 9927645
[31] 韩建国, 樊奋成, 李枫. 禾本科植物的起源、进化及分布. 植物学报, 1996,1:10-14.
Han J G, Fan F C, Li F. Origin, evolution and distribution of the Gramineae. Chin Bull Bot, 1996,1:10-14 (in Chinese with English abstract).
[32] Yu J, Meng Z, Liang W, Behera S, Jörg K, Tucker M R, Luo Z, Chen M, Xu D, Zhao G, Wang J, Zhang S, Kim Y J, Zhang D. A rice Ca2+ binding protein is required for tapetum function and pollen formation. Plant Physiol, 2016,176:1772-1786.
[33] Kapoor S. Silencing of the tapetum-specific zinc finger gene TAZ1 causes premature degeneration of tapetum and pollen abortion in Petunia. Plant Cell, 2002,14:2353-2367.
doi: 10.1105/tpc.003061
[34] Shi Y, Zhao S, Yao J. Premature tapetum degeneration: a major cause of abortive pollen development in photoperiod sensitive genic male sterility in rice. J Integr Plant Biol, 2010,51:774-781.
doi: 10.1111/jipb.2009.51.issue-8
[1] 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.
[2] 吴然然, 林云, 陈景斌, 薛晨晨, 袁星星, 闫强, 高营, 李灵慧, 张勤雪, 陈新. 绿豆雄性不育突变体msm2015-1的遗传学与细胞学分析[J]. 作物学报, 2021, 47(5): 860-868.
[3] 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.
[4] 李京琳, 李佳林, 李新鹏, 安保光, 曾翔, 吴永忠, 黄培劲, 龙湍. 水稻ptc1隐性核不育系的创制及其配合力分析[J]. 作物学报, 2021, 47(11): 2173-2183.
[5] 陈淼, 谢赛, 王超智, 李焱龙, 张献龙, 闵玲. 棉花GhPIF4调控高温下花药败育机制初探[J]. 作物学报, 2020, 46(9): 1368-1379.
[6] 蒋会兵,杨盛美,刘玉飞,田易萍,孙云南,陈林波,唐一春. 厚轴茶雄性不育株花药败育的生物学特性和细胞学研究[J]. 作物学报, 2020, 46(7): 1076-1086.
[7] 田士可, 秦心儿, 张文亮, 董雪, 代明球, 岳兵. 玉米雄性不育突变体mi-ms-3的遗传分析及分子鉴定[J]. 作物学报, 2020, 46(12): 1991-1996.
[8] 牟碧涛,赵卓凡,岳灵,李川,张钧,李章波,申汉,曹墨菊. 两份玉米CMS-C恢复系的育性恢复力测定及恢复基因的分子标记定位[J]. 作物学报, 2019, 45(2): 225-234.
[9] 王晓娟,潘振远,刘敏,刘忠祥,周玉乾,何海军,邱法展. 一个新的玉米silky1基因等位突变体的遗传分析与分子鉴定[J]. 作物学报, 2019, 45(11): 1649-1655.
[10] 张伟, 尹米琦, 赵佩, 王轲, 杜丽璞, 叶兴国. 我国部分主推小麦品种组织培养再生能力评价[J]. 作物学报, 2018, 44(02): 208-217.
[11] 孙辉**,张风廷**,王永波,叶志杰,秦志列,白秀成,杨吉芳,高建刚,赵昌平*. 雄性不育小麦BS210育性转换特性[J]. 作物学报, 2017, 43(02): 171-178.
[12] 陶瑶，王瑜，钟思荣，吴凌敏，谢丽娟，聂亚平，周玮，王建革，刘齐元. 烟草ATP合酶F0部分4个亚基基因转录本编辑位点分析[J]. 作物学报, 2016, 42(12): 1743-1753.
[13] 王亚梁**,张玉屏**,朱德峰*,向镜,武辉,陈惠哲,张义凯. 水稻穗分化期高温胁迫对颖花退化及籽粒充实的影响[J]. 作物学报, 2016, 42(09): 1402-1410.
[14] 张小军，岳福良，张小红，侯睿，张相琼，李文均. 利用宿根进行棉花不育系制种的技术体系研究[J]. 作物学报, 2015, 41(12): 1836-1843.
[15] 王震,范晓静,张淼,张芳凝,李桂东,马翎健*. ATP合成相关基因在小麦BNS不育系育性转换中的差异表达[J]. 作物学报, 2014, 40(08): 1501-1505.
Viewed
Full text


Abstract

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