刘彦英;倪珊珊;项蕾蕾;孙雪丽;陈裕坤;赖钟雄;

• 1:福建农林大学园艺植物生物工程研究所

摘要(Abstract)：

【目的】研究香蕉中miR408b及其靶基因Chemocyanin在低温胁迫下的表达模式和调控机制。【方法】以三明野生蕉和‘天宝蕉’叶片为材料,利用RT-PCR和RACE技术克隆获得三明野生蕉中的MiChemocyanin基因(MiCHY)和‘天宝蕉’中的MaChemocyanin基因(MaCHY),利用在线网站对MiCHY进行生物信息学分析。【结果】MiCHY基因的cDNA全长为587 bp,开放阅读框381 bp,编码126个氨基酸蛋白,具有Plantacyanin结构域,属于超铜氧化还原蛋白家族。在ORF区的4～24 bp处有一处高度匹配miR408b的结合区(GCTCAGGGAAGGGGCAGTGCG)。RLM-5’RACE分析表明,miR408b主要在靶序列的第7～8个碱基之间剪切靶基因MiCHY的mRNA。序列比对发现,MaCHY基因比MiCHY基因多了82 bp的序列,符合内含子的GT-AG剪切,推测发生了内含子驻留的可变剪接。保守结构域分析表明MaCHY没有Plantacyanin结构域,但也属于铜氧化还原蛋白家族。qRT-PCR结果表明,低温胁迫下,冷敏感的‘天宝蕉’miR408b的表达量上升,MaCHY的表达量降低;在抗寒的三明野生蕉中,miR408b的表达量下降,MiCHY表达量上升,miR408b与MiCHY表达量呈负相关。【结论】miR408b及其靶基因MiCHY可能在香蕉响应低温胁迫中发挥重要作用。

关键词(KeyWords)： 香蕉;miR408b;Chemocyanin基因;低温胁迫;表达分析

Abstract:

Keywords:

基金项目(Foundation): 国家重点研发计划(2019YFD1000901);; 国家现代农业产业技术体系(香蕉)专项资金(GARS-31-15);; 福建省高原学科建设经费(102/71201801101);; 福建农林大学科技创新专项基金(CXZX2018076)

作者(Author): 刘彦英;倪珊珊;项蕾蕾;孙雪丽;陈裕坤;赖钟雄;

Email:

参考文献(References)：

• [1] SUNKAR R. MicroRNAs with macro-effects on plant stress responses[J]. Seminars in Cell&Developmental Biology,2010,21(8):805-811.
• [2] JONES-RHOADES M W,BARTEL D P,BARTEL B. MicroRNAS and their regulatory roles in plants[J]. Annual Review of Plant Biology,2006,57(1):19-53.
• [3] SUNKAR R,ZHU J K. Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis[J]. The Plant Cell,2004,16(8):2001-2019.
• [4] LU S F,SUN Y H,SHI R,CLARK C,LI L G,CHIANG V L.Novel and mechanical stress-responsive microRNAs in Populus trichocarpa that are absent from Arabidopsis[J]. Plant Cell,2005,17(8):2186-2203.
• [5] TRINDADE I,CAPIT?O C,DALMAY T,FEVEREIRO M P,SANTOS DMD. miR398 and miR408 are up-regulated in response towater deficit in Medicago truncatula[J]. Planta,2010,231(3):705-716.
• [6] KANTAR M,UNVER T,BUDAK H. Regulation of Barley miRNAs upon dehydration stress correlated with target gene expression[J]. Functional&Integrative Genomics,2010,10(4):493-507.
• [7] ELDEM V，?ELIKKOL AK?AY U,OZHUNER E,BAK?R Y,URANBEY S,UNVER T. Genome-wide identification of miRNA sresponsive to drought in peach(Prunus persica)by high-throughput deep sequencing[J]. PLoS One,2012,7(12):e50298.
• [8] OZHUNER E,ELDEM V,IPEK A,SAKCALI S. ZHANG B,BOKE H,UNVER T. Boron stress responsive microRNAs and their targets in barley[J]. PLoS One,2013,8(3):e59543.
• [9] ZHOU L G,LIU Y H,LIU Z C,KONG D Y,DUAN M,LUO L J. Genome-wide identification and analysis of drought-responsive microRNAs in Oryza sativa[J]. Journal of Experimental Botany,2010,61(15):4157-4168.
• [10] MA C,BURD S,LERS A. miR408 is involved in abiotic stress responses in Arabidopsis[J]. The Plant Journal,2015,84(1):169-187.
• [11] HAJYZADEH M,TURKTAS M K,UNVER T. miR408 overexpression causes increased drought tolerance in chickpea[J].Gene,2015,555(2):186-193.
• [12] PAN J W,HUANG D H,GUO Z L,KUANG Z,ZHANG H,XIE X Y,MA Z F,GAO S P,LERDAU M T,CHU C C,LI L. Overexpression of icroRNA408 enhances photosynthesis,growth,and seed yield in diverse plants[J]. Journal of Integrative Plant Biology,2018,60(4):323-340.
• [13]马圣运. Os-miR408的表达模式及其在水稻种子发育中的功能[D].杭州：浙江大学，2012.MA Shengyuan. Expression pattern of Os-miR408 and its function in rice seed development[D]. Hangzhou:Zhejiang University,2012.
• [14] FENG H,ZHANG Q,WANG Q L,WANG X J,LIU J,LI M,HUANG L L,KANG Z S. Target of tae-miR408,a chemocyanin-like protein gene(TaCLP1),plays positive roles in wheat response to high-salinity,heavy cupric stress and stripe rust[J]. Plant Molecular Biology,2013,83(4/5):433-443.
• [15] RYDéN,L G,HUNT L T. Evolution of protein complexity:the blue copper-containing oxidases and related proteins[J]. Journal of Molecular Evolution,1993,36(1):41-66.
• [16] KIM S,MOLLET J C,DONG J,ZHANG K L,PARK S Y,LORD E M. Chemocyanin,a small basic protein from the lily stigma,induces pollen tube chemotropism[J]. Proceedings of the National Academy of Sciences of the United States of America,2003,100(26):16125-16130.
• [17] RICHARDS K D,SCHOTT E J,SHARMA Y K,DAVIS K R,GARDNER R C. Aluminum induces oxidative stress genes in Arabidopsis thaliana[J]. Plant Physiology,1998,116(1):409-418.
• [18] MA H L,ZHAO H M,LIU Z,ZHAO J. The phytocyanin gene family in rice(Oryza sativa L.):genome-wide identification,classification and transcriptional analysis[J]. PLoS One,2011,6(10):e25184.
• [19] RUAN X M,LUO F,LI D D,ZHANG J,LIU Z H,XU W L,HUANG G Q,LI X B. Cotton BCP genes encoding putative blue copper-binding proteins are functionally expressed in fiber development and involved in response to high-salinity and heavy metal stresses[J]. Physiologia Plantarum,2011,141(1):71-83.
• [20]刘炜婳.基于全转录组学的野生蕉（Musa itinerans）低温胁迫响应机制研究[D].福州：福建农林大学，2018.LIU Weihua. Studies on the responsive mechanism of wild banana(Musa itinerans)to low-temperature stress based on the transcription of RNA-seq technology[D]. Fuzhou:Fujian Agriculture and Forestry University,2018.
• [21]金安娜，李建雄，田志宏.植物中miRNA及其靶基因mRNA剪切位点的常用检测方法[J].长江大学学报(自然科学版)，2014,11(11):43-50.JING Anna,LI Jianxiong,TIAN Zhihong. Common detection method for siRNA and its target gene mRNA cleavage site in plant[J]. Journal of Yangtze University(Natural Science Edition),2014,11(11):43-50.
• [22] JOVANOVI??，STANISAVLJEVI?A N,MIKI?B A,RADOVI?C S,MAKSIMOVI?A V. Water deficit down-regulates miR398and miR408 in pea(Pisum sativum L.)[J]. Plant Physiology&Biochemistry,2014,83:26-31.
• [23]李利超，孙化雨，杨意宏，赵韩生，王思宁，高志民.毛竹miR398和miR408的克隆及其表达分析[J].热带亚热带植物学报，2017,25(3):241-249.LI Lichao,SUN Huayu,YANG Yihong,ZHAO Hansheng,WANG Sining,GAO Zhimin. Cloning and expression analysis of Phyllostachys pubescens miR398 and miR408[J]. Journal of Tropical and Subtropical Botany,2017,25(3):241-249.
• [24]郭晓荣.丹参miR408前体序列的克隆及功能分析[D].西安：陕西师范大学，2017.GUO Xiaorong. Cloning and functional analysis of the precursor sequence of Salvia miR408[D]. Xi’an:Shanxi Normal University,2017.
• [25] PE?ARRUBIA L,ROMERO P,CARRIó-SEGUíA,ANDRéS-BORDERíA A,MORENO J,SANZ A. Temporal aspects of copper homeostasis and its crosstalk with hormones[J].Frontiers in Plant Science,2015,6:255.
• [26] SEO P J,PARK M J,PARK C M. Alternative splicing of transcription factors in plant responses to low temperature stress:mechanisms and functions[J]. Planta,2013,237(6):1415-1424.
• [27]熊雪梅.盐芥冷响应miRNA表达模式分析及tsa-miR396功能初探[D].哈尔滨：东北林业大学，2014.XIONG Xuemei. Cold-response miRNA expression pattern analysis and tsa-miR396 function in Thellungiella salsuginea[D].Harbin:Northeast Forestry University,2014.
• [28] ZHANG F,ZHANG Y C,ZHANG J P,YU Y,ZHOU Y F,FENG Y Z. Rice UCL8,a plantacyanin gene targeted by miR408,regulates fertility by controlling pollen tube germination and growth[J]. Rice,2018,11(1):60.
• [29] YANG Y T,ZHANG X,SU Y C,ZOU J K,WANG Z T,XU L P,QUE Y X. miRNA alteration is an important mechanism in sugarcane response to low-temperature environment[J]. BMC Genomics,2017,18(1):833.
• [30] RYDéN L G,HUNT L T. Evolution of protein complexity:The blue copper-containing oxidases and related proteins[J]. Journal of Molecular Evolution,1993,36(1):41-66.
• [31]姜丽丽，邸宏，林忠平，胡鸢雷，吴韩英，崔少彬，姜丽静，卢翠华.蓝铜蛋白类似基因BcBCP1转化马铃薯及其抗旱性的改良[J].中国蔬菜，2009(2):7-11.JIANG Lili,DI Hong,LIN Zhongping,HU Yuanlei,WU Hanying,CUI Shaobin,JIANG Lijing,LU Cuihua. Transformation of potato with brassin like gene BcBCP1 and improvement of its drought resistance[J]. Chinese Vegetables,2009(2):7-11.
• [32] UEDA T,NOMOTO N,KOGA M,OGASA K,OGAWA Y,MATSUMOTO M,STAMPOULIS P,SODE K,TERASAWA H,SHIMADA I. Structural basis of efficient electron transport between photosynthetic membrane proteins and plastocyanin in spinach revealed using nuclear magnetic resonance[J]. Plant Cell,2012,24(10):4173-4186.
• [33] SIGFRIDSSON K. Plastocyanin,an electron-transfer protein[J].Photosynthesis Research,1998,57(1):1-28.
• [34] MITTLER R. ROS and redox signalling in the response of plants to abiotic stress[J]. Plant Cell&Environment,2012,35(2):259-270.
• [35] OELZE M L,KANDLBINDER A,DIETZ KJ. Redox regulation and overreduction control in the photosynthesizing cell:complexity in redox regulatory networks[J]. Biochimica Et Biophysica Acta General Subjects,2008,1780(11):1261-1272.
• [36] JANMOHAMMADI M,ZOLLA L,RINALDUCCI S. Low temperature tolerance in plants:Changes at the protein level[J].Phytochemistry,2015,117:76-89.
• [37] ZHANG J P,YU Y,FENG Y Z,ZHOU Y F,ZHANG F,YANG Y W,LEI M Q,ZHANG Y C,CHEN Y Q. MiR408regulates grain yield and photosynthesis via a phytocyanin protein[J]. Plant Physiology,2017,175(3):1175.