龙眼miR397家族成员分子特性及其在体胚发生早期的表达分析
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摘要
【目的】探讨龙眼miR397家族成员的分子特性及其在龙眼体胚发生早期的表达模式。【方法】以‘红核子’龙眼胚性愈伤组织为材料,通过对龙眼miR397前体序列二级结构分析、进化树构建、靶基因预测与裂解位点验证、以及龙眼miR397a在龙眼体胚发育早期和不同激素浓度处理下的表达规律进行分析。【结果】龙眼miR397前体序列5端臂上可生成两个序列高度重合的成熟体序列dlo-miR397a和dlo-miR397,二者仅有5端两个碱基TC之间的差异,前体能形成稳定的茎环结构;进化树分析发现龙眼miR397前体与桃、可可、脐橙等亲缘关系较近,处于同一分支,龙眼miR397与拟南芥、甜橙、葡萄等亲缘关系较近,处于较为保守的同一分支,miR397家族成员较前体序列在进化上更为保守。靶基因预测表明龙眼miR397家族主要靶向调控漆酶基因家族和一条姐妹染色单体粘连蛋白(dlo_039206.1)。裂解位点验证发现靶基因姐妹染色单体粘连蛋白的裂解位点,但位于预测区间外。miR397a在体胚发生过程中对于靶基因姐妹染色单体粘连蛋白的调控模式在0～3 d较为一致,均为下调表达,6～12 d为显著上调表达,且二者呈典型的负调控关系。龙眼miR397a在不同浓度2,4-D与GA3激素处理下均出现下调表达趋势,而靶基因姐妹染色单体粘连蛋白则在激素处理下的表达趋势并不明显,可能由于靶向裂解位点在预测区外所致。【结论】龙眼miR397a可能在龙眼体胚发生早期的6～12 d,通过靶向调控姐妹染色单体粘连蛋白影响龙眼体胚发生早期形态建成。龙眼miR397家族在进化上较为保守,其靶基因主要靶向调控龙眼漆酶家族和姐妹染色单体粘连蛋白。龙眼miR397a在不同浓度2,4-D与GA3激素处理下均出现下调表达趋势,表明其在龙眼体胚发生早期可能通过激素信号传导参与龙眼体胚发生早期形态建成。
【Objective】Longan(Dimocarpus longan Lour.) is one of the important economic fruit trees in China's subtropical zone. The longan somatic embryogenesis system is a model system for the study of embryogenesis in woody fruit trees. It has been widely used in molecular biology of somatic embryogenesis. miRNA is one of the important factors to influence epigenetic growth and development of the plant. The regulation mechanism in the process of embryogenesis needs further exploration. miR397 is a kind of miRNA that is more conservative in monocotyledon and dicotyledon. Although it has been studied in other plants, the mechanism of action in the process of somatic embryogenesis in longan has not been systematically analyzed. Therefore, longan miR397 precursor molecular characterization and evolutionary analysis of mature members, and its expression analysis at different stages of early longan somatic embryogenesis and different hormone concentrations, can help to fully understand the function of longan miR397 family members in the early stage of longan somatic embryogenesis.【Methods】‘Honghezi'longan embryogenic calli were used as materials, Mfold was used to analyze the secondary structure of the longan miR397 precursor sequence, and PMTED was used to predict the target gene of miR397, MEGA 7.0 was used for the construction of phylogenetic tree of a total of 53 mature bodies.43 precursor sequence was constructed separately. The cleavage site was verified by the modified RLMRACE method. The longan embryogenic cells were cultured in MS(sucrose 20 g · L-1, 2,4-D 1.0 mg · L-1)liquid medium for 1 to 12 d, and 2,4-D and GA3 of different concentration gradients were used to treat the longan embryogenic calli, and the expression of miR397 family members at different early stages of longan somatic embryo development and different hormone concentrations were discussed.【Results】The mature sequences miR397 a and miR397 of the longan pre-miR397 could be generated on the 5-arm of the longan miR397 precursor sequence. There was only 2 bases TC difference at the 5'between them, and the precursor could form a stable stem loop. The phylogenetic tree analysis revealed that the longan pre-miR397 was closely related to peach(ppe), cocoa(tcc), and navel orange(csi) on the same branch. Longan miR397 and Arabidopsis(ath), sweet orange(csi), Grapes(vvi) were closely related,and they were located on the same conservative branch. The miR397 family members were more conservative than the precursor sequences. Target gene predictions indicated that dlo-miR397 family mainly targeted the laccase gene family and a sister chromosome binding protein(dlo_039206.1), miR397 a negatively regulated target gene dlo_039206.1 in the process of somatic embryogenesis to the late stage.Longan miR397 a was down regulated under the treatments of different concentrations of 2,4-D and GA3 hormones. The target gene sister chromatinin(dlo_039206.1) was not significantly expressed under hormone treatments. However, the expression trend of sister chromatin(dlo_039206.1) of target gene was not obvious under hormone treatments, which might be due to the target cleavage site outside the predicted region.【Conclusion】The targeted cleavage site was verified. miR397 a was down regulated expression under the treatments of different concentration of 2,4-D and GA3 hormones, that might response to the auxin and cytokinin signaling.
【Objective】Longan(Dimocarpus longan Lour.) is one of the important economic fruit trees in China's subtropical zone. The longan somatic embryogenesis system is a model system for the study of embryogenesis in woody fruit trees. It has been widely used in molecular biology of somatic embryogenesis. miRNA is one of the important factors to influence epigenetic growth and development of the plant. The regulation mechanism in the process of embryogenesis needs further exploration. miR397 is a kind of miRNA that is more conservative in monocotyledon and dicotyledon. Although it has been studied in other plants, the mechanism of action in the process of somatic embryogenesis in longan has not been systematically analyzed. Therefore, longan miR397 precursor molecular characterization and evolutionary analysis of mature members, and its expression analysis at different stages of early longan somatic embryogenesis and different hormone concentrations, can help to fully understand the function of longan miR397 family members in the early stage of longan somatic embryogenesis.【Methods】‘Honghezi'longan embryogenic calli were used as materials, Mfold was used to analyze the secondary structure of the longan miR397 precursor sequence, and PMTED was used to predict the target gene of miR397, MEGA 7.0 was used for the construction of phylogenetic tree of a total of 53 mature bodies.43 precursor sequence was constructed separately. The cleavage site was verified by the modified RLMRACE method. The longan embryogenic cells were cultured in MS(sucrose 20 g · L-1, 2,4-D 1.0 mg · L-1)liquid medium for 1 to 12 d, and 2,4-D and GA3 of different concentration gradients were used to treat the longan embryogenic calli, and the expression of miR397 family members at different early stages of longan somatic embryo development and different hormone concentrations were discussed.【Results】The mature sequences miR397 a and miR397 of the longan pre-miR397 could be generated on the 5-arm of the longan miR397 precursor sequence. There was only 2 bases TC difference at the 5'between them, and the precursor could form a stable stem loop. The phylogenetic tree analysis revealed that the longan pre-miR397 was closely related to peach(ppe), cocoa(tcc), and navel orange(csi) on the same branch. Longan miR397 and Arabidopsis(ath), sweet orange(csi), Grapes(vvi) were closely related,and they were located on the same conservative branch. The miR397 family members were more conservative than the precursor sequences. Target gene predictions indicated that dlo-miR397 family mainly targeted the laccase gene family and a sister chromosome binding protein(dlo_039206.1), miR397 a negatively regulated target gene dlo_039206.1 in the process of somatic embryogenesis to the late stage.Longan miR397 a was down regulated under the treatments of different concentrations of 2,4-D and GA3 hormones. The target gene sister chromatinin(dlo_039206.1) was not significantly expressed under hormone treatments. However, the expression trend of sister chromatin(dlo_039206.1) of target gene was not obvious under hormone treatments, which might be due to the target cleavage site outside the predicted region.【Conclusion】The targeted cleavage site was verified. miR397 a was down regulated expression under the treatments of different concentration of 2,4-D and GA3 hormones, that might response to the auxin and cytokinin signaling.
引文
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[6]XUE C,YAO J L,QIN M F,ZHANG M Y,ALLAN A C,WANG D F,WU J.PbrmiR397a regulates lignification during stone cell development in pear fruit[J].Plant Biotechnology Journal,2019,17(1):103-117.
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[11]徐小萍,陈晓慧,吕科良,陈旭,陈裕坤,林玉玲,赖钟雄.龙眼漆酶家族成员全基因组结构与功能分析[J].应用与环境生物学报,2018,24(4):833-844.XU Xiaoping,CHEN Xiaohui,LüKeliang,CHEN Xu,CHEN Yukun,LIN Yuling,LAI Zhongxiong.Genome-wide identifcation and function analysis of the laccase gene family in Dimocarpus longan Lour.[J].Journal of Applied and Environmental Biology,2018,24(4):833-844.
[12]LI H Y,ZHANG J,YANG Y,JIA N N,WANG C X,SUN HM.miR171 and its target gene SCL6 contribute to embryogenic callus induction and torpedo-shaped embryo formation during somatic embryogenesis in two lily species[J].Plant Cell Tissue&Organ Culture,2017,130(3):591-600.
[13]LIN Y,LAI Z.Comparative analysis reveals dynamic changes in miRNAs and their targets and expression during somatic embryogenesis in longan(Dimocarpus longan Lour.)[J].Plos One,2013,8(4):e60337.
[14]曾友竞.miPEPs在龙眼体胚发生过程中的应用研究[D].福州:福建农林大学,2017.ZENG Youjing.Application of miPEPs in somatic embryogenesis of Dimocarpus longan Lour.[D].Fuzhou:University of Agriculture and Forestry in Fujian,2017.
[15]赖钟雄,陈振光.龙眼胚性愈伤组织的高频率体细胞胚胎发生[J].福建农林大学学报(自然科学版),1997,26(3):271-276.LAI Zhongxiong,CHEN Zhenguang.Somatic embryogenesis of high frequency from longan embryogenic calli[J].Journal of Fujian Agricultural ang Forestry University(Natural Science Edition),1997,26(3):271-276.
[16]赖瑞联,林玉玲,赖钟雄.龙眼生长素受体基因TIR1的克隆及其与miR393互作关系[J].应用与环境生物学报,2016,22(1):95-102.LAI Ruilian,LIN Yuling,LAI Zhongxiong.Cloning of auxin receptor gene TIR1 and its interaction with miR393 in Dimocarpus longan Lour.[J].Chinese Journal of Applied&Environmental Biology,2016,22(1):95-102.
[17]王亚婷.龙眼胚性愈伤组织中若干microRNA的初级体克隆及其功能分析[D].福州:福建农林大学,2013.WANG Yating.Studies on cloning of the primary transcripts and function analyses of some microRNAs from embryonic callus in Dimocarpus longan Lour.[D].Fuzhou:University of Agriculture and Forestry in Fujian,2013.
[18]林玉玲.龙眼体胚发生过程中SOD基因家族的克隆及表达调控研究[D].福州:福建农林大学,2011.LIN Yuling.Studies on cloning,expression and regulation of SOD gene family during somatic embryogenesis in Dimocarpus longan Lour.[D].Fuzhou:University of Agriculture and Forestry in Fujian,2011.
[19]赵守城,朱壮春,赵亚龙.姐妹染色单体间的粘连与粘连蛋白[J].生物学教学,2004,29(7):7-8.ZHAO Shoucheng,ZHU Zhuangchun,ZHAO Yalong.Adhesion and Adhesion protein between sister chromatids[J].Biology Teaching,2004,29(7):7-8.
[20]董园园,刘秀明,姚娜,赵利旦,李海燕.红花miR397a基因表达及对靶基因LAC2的调控作用[J].西北农林科技大学学报(自然科学版),2016,44(7):173-180.DONG Yuanyuan,LIU Xiuming,YAO Na,ZHAO Lidan,LIHaiyan,Expression of safflower miR397a gene and its role in LAC2 regulation.[J].Journal of Northwestern University of Agriculture and Forestry Science and Technology(Natural Science Edition),2016,44(7):173-180.
[21]李利超,孙化雨,娄永峰,杨意宏,赵韩生,高志民.毛竹漆酶基因PeLAC的克隆与表达分析[J].植物科学学报,2017,35(2):252-259.LI Lichao,SUN Huayu,LOU Yongfeng,YANG Yihong,ZHAO Hansheng,GAO Zhimin.Cloning and expression analysis of PeLAC in phyllostachys edulis[J].Journal of Plant Science,2017,35(2):252-259.
[22]QIANG X,LIU Y,ZHU A,WU X M,YE J L,YU K Q,GUOW W,DENG X X.Discovery and comparative profiling of microRNAs in a sweet orange red-flesh mutant and its wild type[J].BMC Genomics,2010,11(1):246.
[23]WU X M,LIU M Y,GE X X,XU Q,GUO W W.Stage and tissue-specific modulation of ten conserved miRNAs and their targets during somatic embryogenesis of Valencia sweet orange[J].Planta,2011,233(3):495-505.
[2]DAI X,ZHUANG Z,ZHAO P X.Computational analysis of miRNA targets in plants:current status and challenges[J].Briefings in Bioinformatics,2011,12(2):115-121.
[3]GAIDATZIS D,NIMWEGEN E V,HAUSSER J,ZAVOLANM.Inference of miRNA targets using evolutionary conservation and pathway analysis[J].BMC Bioinformatics,2007,8(1):1-22.
[4]DONG C H,PEI H.Over-expression of miR397 improves plant tolerance to cold stress in Arabidopsis thaliana[J].Journal of Plant Biology,2014,57(4):209-217.
[5]XIANG J,LIN P,LI X S,LIU M Y,ZHANG L,GUO Y D.Overexpression of tomato Sly-miR397 gene enhances drought tolerance in Arabidopsis thaliana[J].Journal of China Agricultural University,2016,21(10):51-58.
[6]XUE C,YAO J L,QIN M F,ZHANG M Y,ALLAN A C,WANG D F,WU J.PbrmiR397a regulates lignification during stone cell development in pear fruit[J].Plant Biotechnology Journal,2019,17(1):103-117.
[7]HUANG J H,QI Y P,WEN S X,GUO P,CHEN X M,CHEN L S.Illumina microRNA profiles reveal the involvement of miR397a in Citrus adaptation to long-term boron toxicity via modulating secondary cell-wall biosynthesis[J].Scientific Reports,2016,6:22900.
[8]ZHANG Y,ZHANG S G,HAN S Y,LI X M,QI L W.Transcriptome profiling and in silico analysis of somatic embryos in Japanese larch(Larix leptolepis)[J].Plant Cell Reports,2012,31(9):1637-1657.
[9]ZHANG Y C,YU Y,WANG C Y,LI Z Y,LIU Q,XU J,LI-AO J Y,WANG X J,QU L H,CHEN F.Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching[J].Nature Biotechnology,2013,31(9):848-852.
[10]PAN L,ZHAO H W,YU Q,BAI L Y,DONG L Y.miR397/laccase gene mediated network improves tolerance to fenoxaprop-P-ethyl in beckmannia syzigachne and Oryza sativa[J].Frontiers in Plant Science,2017,8:879.
[11]徐小萍,陈晓慧,吕科良,陈旭,陈裕坤,林玉玲,赖钟雄.龙眼漆酶家族成员全基因组结构与功能分析[J].应用与环境生物学报,2018,24(4):833-844.XU Xiaoping,CHEN Xiaohui,LüKeliang,CHEN Xu,CHEN Yukun,LIN Yuling,LAI Zhongxiong.Genome-wide identifcation and function analysis of the laccase gene family in Dimocarpus longan Lour.[J].Journal of Applied and Environmental Biology,2018,24(4):833-844.
[12]LI H Y,ZHANG J,YANG Y,JIA N N,WANG C X,SUN HM.miR171 and its target gene SCL6 contribute to embryogenic callus induction and torpedo-shaped embryo formation during somatic embryogenesis in two lily species[J].Plant Cell Tissue&Organ Culture,2017,130(3):591-600.
[13]LIN Y,LAI Z.Comparative analysis reveals dynamic changes in miRNAs and their targets and expression during somatic embryogenesis in longan(Dimocarpus longan Lour.)[J].Plos One,2013,8(4):e60337.
[14]曾友竞.miPEPs在龙眼体胚发生过程中的应用研究[D].福州:福建农林大学,2017.ZENG Youjing.Application of miPEPs in somatic embryogenesis of Dimocarpus longan Lour.[D].Fuzhou:University of Agriculture and Forestry in Fujian,2017.
[15]赖钟雄,陈振光.龙眼胚性愈伤组织的高频率体细胞胚胎发生[J].福建农林大学学报(自然科学版),1997,26(3):271-276.LAI Zhongxiong,CHEN Zhenguang.Somatic embryogenesis of high frequency from longan embryogenic calli[J].Journal of Fujian Agricultural ang Forestry University(Natural Science Edition),1997,26(3):271-276.
[16]赖瑞联,林玉玲,赖钟雄.龙眼生长素受体基因TIR1的克隆及其与miR393互作关系[J].应用与环境生物学报,2016,22(1):95-102.LAI Ruilian,LIN Yuling,LAI Zhongxiong.Cloning of auxin receptor gene TIR1 and its interaction with miR393 in Dimocarpus longan Lour.[J].Chinese Journal of Applied&Environmental Biology,2016,22(1):95-102.
[17]王亚婷.龙眼胚性愈伤组织中若干microRNA的初级体克隆及其功能分析[D].福州:福建农林大学,2013.WANG Yating.Studies on cloning of the primary transcripts and function analyses of some microRNAs from embryonic callus in Dimocarpus longan Lour.[D].Fuzhou:University of Agriculture and Forestry in Fujian,2013.
[18]林玉玲.龙眼体胚发生过程中SOD基因家族的克隆及表达调控研究[D].福州:福建农林大学,2011.LIN Yuling.Studies on cloning,expression and regulation of SOD gene family during somatic embryogenesis in Dimocarpus longan Lour.[D].Fuzhou:University of Agriculture and Forestry in Fujian,2011.
[19]赵守城,朱壮春,赵亚龙.姐妹染色单体间的粘连与粘连蛋白[J].生物学教学,2004,29(7):7-8.ZHAO Shoucheng,ZHU Zhuangchun,ZHAO Yalong.Adhesion and Adhesion protein between sister chromatids[J].Biology Teaching,2004,29(7):7-8.
[20]董园园,刘秀明,姚娜,赵利旦,李海燕.红花miR397a基因表达及对靶基因LAC2的调控作用[J].西北农林科技大学学报(自然科学版),2016,44(7):173-180.DONG Yuanyuan,LIU Xiuming,YAO Na,ZHAO Lidan,LIHaiyan,Expression of safflower miR397a gene and its role in LAC2 regulation.[J].Journal of Northwestern University of Agriculture and Forestry Science and Technology(Natural Science Edition),2016,44(7):173-180.
[21]李利超,孙化雨,娄永峰,杨意宏,赵韩生,高志民.毛竹漆酶基因PeLAC的克隆与表达分析[J].植物科学学报,2017,35(2):252-259.LI Lichao,SUN Huayu,LOU Yongfeng,YANG Yihong,ZHAO Hansheng,GAO Zhimin.Cloning and expression analysis of PeLAC in phyllostachys edulis[J].Journal of Plant Science,2017,35(2):252-259.
[22]QIANG X,LIU Y,ZHU A,WU X M,YE J L,YU K Q,GUOW W,DENG X X.Discovery and comparative profiling of microRNAs in a sweet orange red-flesh mutant and its wild type[J].BMC Genomics,2010,11(1):246.
[23]WU X M,LIU M Y,GE X X,XU Q,GUO W W.Stage and tissue-specific modulation of ten conserved miRNAs and their targets during somatic embryogenesis of Valencia sweet orange[J].Planta,2011,233(3):495-505.
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