龙须菜四分孢子体不同发育阶段差异表达基因克隆和启动子区分析与刺激响应研究
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摘要
龙须菜四分孢子体形成的四分孢子囊数量和四分孢子放散量可以作为育性高低的指标。龙须菜品种"981"在成熟期四分孢子放散量极低,与"鲁龙1号"有显著差异,即"981"具有低育的育性特点。从2个品种不同发育阶段数字表达谱数据中,筛选得到与育性控制相关的差异表达基因——D-乳酸脱氢酶(dld),谷氨酰胺合成酶(gln2)和热激蛋白(hsp15.4)。克隆得到dld完整的ORF与上游序列并进行测序分析,设置诱导条件检测3个基因的表达量,探究基因表达的调控因素。dld基因具有D-乳酸脱氢酶结构域、龙须菜特有的2个跨膜区、1个低复杂性区域。启动子区功能预测发现3个基因均可能受光照、茉莉酸甲酯和一些植物激素的调控。通过半定量PCR技术检测不同诱导条件下3个基因的表达变化趋势,结果显示光照与茉莉酸甲酯均可刺激3个基因的正表达。
The number of tetrasporangium formed and tetraspores released by Gracilariopsis lemaneiformis tetrasporophytes could be a judgement standard for the criteria of fertility level.The tetraspores released by cultivar 981 during its maturation is extremely low,which is significantly difference with cultivar No.1 Lulong.Thus 981 is characterized as low fertility.From the DGE data of the two strains under different development stages,significantly differentially expressed genes D-lactate dehydrogenase(dld),glutamine(gln)and hot shock protein(hsp)were screened and regarded as related to fertility control.Partial upstream and complete ORF sequences of dldwere cloned and analyzed.The expression of three genes under different induction conditions was detected to explore the gene regulation mechanism.The dldgene had one D-lactate dehydrogenase domain,two transmembrane regions which are unique to G.lemaneiformis;and one low complexity region.Promoter analysis in the upstream region showed that the expression of three genes might be regulated by light,methy jasmonate and some plant hormones.Semiquantitative RT-PCR was operated to detect gene expression under different induction conditions.Results showed that the expression level of three genes had an obviously positive correlation with light and methy jasmonate treatment.
The number of tetrasporangium formed and tetraspores released by Gracilariopsis lemaneiformis tetrasporophytes could be a judgement standard for the criteria of fertility level.The tetraspores released by cultivar 981 during its maturation is extremely low,which is significantly difference with cultivar No.1 Lulong.Thus 981 is characterized as low fertility.From the DGE data of the two strains under different development stages,significantly differentially expressed genes D-lactate dehydrogenase(dld),glutamine(gln)and hot shock protein(hsp)were screened and regarded as related to fertility control.Partial upstream and complete ORF sequences of dldwere cloned and analyzed.The expression of three genes under different induction conditions was detected to explore the gene regulation mechanism.The dldgene had one D-lactate dehydrogenase domain,two transmembrane regions which are unique to G.lemaneiformis;and one low complexity region.Promoter analysis in the upstream region showed that the expression of three genes might be regulated by light,methy jasmonate and some plant hormones.Semiquantitative RT-PCR was operated to detect gene expression under different induction conditions.Results showed that the expression level of three genes had an obviously positive correlation with light and methy jasmonate treatment.
引文
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[11]蔡红梅,肖景华,张启发,练兴明.抑制表达谷氨酰胺合成酶基因对水稻氮代谢和生长发育的影响[J].科学通报,2010,55(10):871-882.Co-suppressed glutamine synthetase2gene modifies nitrogen metabolism and plant growth in rice.[J]Science Bulletin,2010,55(10):871-882.
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[13]栗振义,龙瑞才,张铁军,等.植物热激蛋白研究进展[J].生物技术通报,2016,32(2):7-13.Li Z Y,Long R C,Zhang T J,et al.Research progress on plant Heat Shock Protein[J].Biotechnology Bulletin,2016,32(2):7-13.
[14]Timperio A M,Egidi M G,Zolla L.Proteomics applied on plant abiotic stresses:Role of heat shock proteins(HSP15.4)[J].Journal of Proteomics,2008,71(4):391-411.
[15]Al-Whaibi M H.Plant heat-shock proteins:A mini review[J].Journal of King Saud University-Science,2011,23(2):139-150.
[16]苏晴,茹振钢,秦志英,等.小热激蛋白基因(hsp23.5)在小麦BNS雄性不育系和转换系中的差异表达[J].农业生物技术学报,2013,21(1):29-37.Su Q,Ru Z G,Qin Z Y,et al.Differential expression of small heat shock protein gene(hsp23.5)between wheat(Triticum aestivum)BNS male sterile line and its conversion line[J].Journal of Agricultural Biotechnology,2013,21(1):29-37.
[17]Zhou W,Sui Z,Wang J,et al.An orthogonal design for optimization of growth conditions for all life history stages of Gracilariopsis lemaneiformis,(Rhodophyta)[J].Aquaculture,2013,s392-395(10):98-105.
[18]Garcia-Jimenez P,Brito-Romano O,Robaina R R.Occurrence of jasmonates during cystocarp development in the red algaGrateloupia imbricata[J].Journal of Phycology,2016,52(6):1085-1093.
[19]Wei Z,Hu Y,Sui Z,et al.Genome survey sequencing and genetic background characterization of Gracilariopsis lemaneiformis(Rhodophyta)based on next-generation sequencing[J].Plos One,2013,8(7):e69909.
[20]杜青伟.龙须菜质体基因组进化分析及红藻琼胶和淀粉合成途径相关基因的克隆及其表达调控的研究[D].青岛:中国海洋大学,2016.Du Q W.Evolutionary Analysis of Chloroplast Genome and Studies on the Gene Cloning and Expressions Regulations Involving in the Pathway of Agar and Floridean Starch Synthesis of Gracilariopsis lemaneiformis(Rhodophyta)[D].Qingdao:Ocean University of China,2016.
[21]彭冲.红藻蜈蚣藻属(Grateloupia C.Agardh)不同地理群体遗传多样性与种群遗传分析及龙须菜(Gracialriopsis lemaneiformis)发育相关DNA甲基化调控研究[D].青岛:中国海洋大学,2016.Peng C.Population Genetic Analysis and Genetic Diversity study in Different Grateloupia Geographical Population and Development Related DNA Metnylation Study in Gracialriopsis lemaneiformis[D].Qingdao:Ocean University of China,2016.
[22]赵建平,姚祥坦.植物小孢子胚胎发生机理[J].生物技术通报,2009,2009(6):7-11.Zhao J P,Yao X T.Mechanism of microspore embryogenesisin plant[J].Biotechnology Bulletin,2009,2009(6):7-11.
[23]荆艳.基于表面及浅层氨基酸特性的胞内蛋白质稳定性分析[D].南京:南京航空航天大学,2012.Jing Y.Computational Analysis of Intracellular Protein Stability Based on Sequence-Derived Features[D].Nanjing:Nanjing University of Aeronautics and Astronautics,2012.
[24]杨予涛.一个光合组织特异表达启动子的克隆、功能分析及其转录因子的鉴定[D].泰安:山东农业大学,2005.Yang Y T.Isolation and Characterization of a Strong Specific Promoter in Photosynthetic Tissues and Identification of a bZIPTranscription Factor[D].Taian:Shandong Agricultural University,2005.
[25]Guzmán-Urióstegui A,Robaina R,Freile-Pelegrín Y,et al.Polyamines increase carpospore output and growth during in vitro cultivation of Hydropuntia cornea[J].Biotechnology Letters,2012,34(4):755-761.
[26]Sacramento A T,García-Jiménez P,Robaina R R.The polyamine spermine induces cystocarp development in the seaweed Grateloupia,(Rhodophyta)[J].Plant Growth Regulation,2007,53(3):147-154.
[27]García-Jiménez P,Robaina R R.Effects of ethylene on tetrasporogenesis in Pterocladiella capillacea(Rhodophyta)[J].Journal of Phycology,2012,48(3):710-715.
[28]Koornneef A,Verhage A,Leon-Reyes A,et al.Towards a reporter system to identify regulators of cross-talk between salicylate and jasmonate signaling pathways in Arabidopsis[J].Plant Signaling&Behavior,2008,3(8):543-546.
[29]Wang F,Wang C,Zou T,et al.Comparative transcriptional profiling of Gracilariopsis lemaneiformisinresponse to salicylic acidand methyl jasmonate-mediated heat resistance[J].Plos One,2017,12(5):e0176531.
[2]Bird C J,Rice E L,Murphy C A,et al.Phylogenetic relationship in the Gracilariales(Rhodophyta)as determined by 18SrDNA sequences[J].Phycologia,1992,31(6):510-522.
[3]Chen W Z,Xu D,Wang L G,et al.Preliminary study on economic characteristics and agar characteristics of two new strains of Gracilaria lemaneiformis[J].Periodical of Ocean University of China,2009.陈伟洲,徐涤,王亮根,等.两个龙须菜新品系经济性状及琼胶特性的初步研究[J].中国海洋大学学报(自然科学版),2009,39(3):437-442.
[4]张学成,秦松,马家海,等.海藻遗传学[M].北京:农业出版社,2005.ZHANG Xue-Cheng,QIN Song,MA Jia-Hai,et al.The Genetics of Marine Algae[M].Beijing:China Agricultural Press,2005.
[5]Kain J M,Destombe C.A review of the life history,reproduction and phenology of Gracilaria[J].Journal of Applied Phycology,1995,7(3):269-281.
[6]王津果.龙须菜四分孢子体发育过程显微结构、生理生化与数字表达谱研究及育性相关性状分析[D].青岛:中国海洋大学,2016.Wang J G.The Study of Microscopic Structure,Physiology,Biochemisty and Digital Expression Profiling During the Development of Tetrasporophytes and the Traits Telated to Fertility of Gracilariopsis lemaneiforis[D].Qingdao:Ocean University of China,2016.
[7]张祐基,杨以勋,王清印,等.条斑紫菜(Porphyra Yezoensis Ueda)遗传和育种的研究[J].海洋湖沼通报,1985(4):46-54.Zhang Y J,Yang Y X,Wang Q Y,et al.Studies on the breeding and genetics of Porphya Yezoensis UEDA[J].Transactions of O-ceanology and Limnology,1985(4):46-54.
[8]Jonsson A.Enumeration and confirmation of clostridium tyrobutyricum,in silages using neutral red,D-Cycloserine,and lactate dehydrogenase activity[J].Journal of Dairy Science,1990,73(3):719-725.
[9]冯万军,邢国芳,牛旭龙,等.植物谷氨酰胺合成酶研究进展及其应用前景[J].生物工程学报,2015,31(9):1301-1312.Feng W J,Xing G F,Niu X L,et al.Progress and application prospects of glutamine synthase in plants[J].Chinese Journal of Biotechnology,2015,31(9):719-725.
[10]Dunbar A J,Wheldrake J F.Evidence for a developmentally regulated prespore-specific glutamine synthetase in the cellular slime mould Dictyostelium discoideum[J].Microbiology,1995,141(Pt 5)(5):1125-1130.
[11]蔡红梅,肖景华,张启发,练兴明.抑制表达谷氨酰胺合成酶基因对水稻氮代谢和生长发育的影响[J].科学通报,2010,55(10):871-882.Co-suppressed glutamine synthetase2gene modifies nitrogen metabolism and plant growth in rice.[J]Science Bulletin,2010,55(10):871-882.
[12]Wang,Wangxia,Vinocur,et al.Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response[J].Trends in Plant Science,2004,9(5):244-252.
[13]栗振义,龙瑞才,张铁军,等.植物热激蛋白研究进展[J].生物技术通报,2016,32(2):7-13.Li Z Y,Long R C,Zhang T J,et al.Research progress on plant Heat Shock Protein[J].Biotechnology Bulletin,2016,32(2):7-13.
[14]Timperio A M,Egidi M G,Zolla L.Proteomics applied on plant abiotic stresses:Role of heat shock proteins(HSP15.4)[J].Journal of Proteomics,2008,71(4):391-411.
[15]Al-Whaibi M H.Plant heat-shock proteins:A mini review[J].Journal of King Saud University-Science,2011,23(2):139-150.
[16]苏晴,茹振钢,秦志英,等.小热激蛋白基因(hsp23.5)在小麦BNS雄性不育系和转换系中的差异表达[J].农业生物技术学报,2013,21(1):29-37.Su Q,Ru Z G,Qin Z Y,et al.Differential expression of small heat shock protein gene(hsp23.5)between wheat(Triticum aestivum)BNS male sterile line and its conversion line[J].Journal of Agricultural Biotechnology,2013,21(1):29-37.
[17]Zhou W,Sui Z,Wang J,et al.An orthogonal design for optimization of growth conditions for all life history stages of Gracilariopsis lemaneiformis,(Rhodophyta)[J].Aquaculture,2013,s392-395(10):98-105.
[18]Garcia-Jimenez P,Brito-Romano O,Robaina R R.Occurrence of jasmonates during cystocarp development in the red algaGrateloupia imbricata[J].Journal of Phycology,2016,52(6):1085-1093.
[19]Wei Z,Hu Y,Sui Z,et al.Genome survey sequencing and genetic background characterization of Gracilariopsis lemaneiformis(Rhodophyta)based on next-generation sequencing[J].Plos One,2013,8(7):e69909.
[20]杜青伟.龙须菜质体基因组进化分析及红藻琼胶和淀粉合成途径相关基因的克隆及其表达调控的研究[D].青岛:中国海洋大学,2016.Du Q W.Evolutionary Analysis of Chloroplast Genome and Studies on the Gene Cloning and Expressions Regulations Involving in the Pathway of Agar and Floridean Starch Synthesis of Gracilariopsis lemaneiformis(Rhodophyta)[D].Qingdao:Ocean University of China,2016.
[21]彭冲.红藻蜈蚣藻属(Grateloupia C.Agardh)不同地理群体遗传多样性与种群遗传分析及龙须菜(Gracialriopsis lemaneiformis)发育相关DNA甲基化调控研究[D].青岛:中国海洋大学,2016.Peng C.Population Genetic Analysis and Genetic Diversity study in Different Grateloupia Geographical Population and Development Related DNA Metnylation Study in Gracialriopsis lemaneiformis[D].Qingdao:Ocean University of China,2016.
[22]赵建平,姚祥坦.植物小孢子胚胎发生机理[J].生物技术通报,2009,2009(6):7-11.Zhao J P,Yao X T.Mechanism of microspore embryogenesisin plant[J].Biotechnology Bulletin,2009,2009(6):7-11.
[23]荆艳.基于表面及浅层氨基酸特性的胞内蛋白质稳定性分析[D].南京:南京航空航天大学,2012.Jing Y.Computational Analysis of Intracellular Protein Stability Based on Sequence-Derived Features[D].Nanjing:Nanjing University of Aeronautics and Astronautics,2012.
[24]杨予涛.一个光合组织特异表达启动子的克隆、功能分析及其转录因子的鉴定[D].泰安:山东农业大学,2005.Yang Y T.Isolation and Characterization of a Strong Specific Promoter in Photosynthetic Tissues and Identification of a bZIPTranscription Factor[D].Taian:Shandong Agricultural University,2005.
[25]Guzmán-Urióstegui A,Robaina R,Freile-Pelegrín Y,et al.Polyamines increase carpospore output and growth during in vitro cultivation of Hydropuntia cornea[J].Biotechnology Letters,2012,34(4):755-761.
[26]Sacramento A T,García-Jiménez P,Robaina R R.The polyamine spermine induces cystocarp development in the seaweed Grateloupia,(Rhodophyta)[J].Plant Growth Regulation,2007,53(3):147-154.
[27]García-Jiménez P,Robaina R R.Effects of ethylene on tetrasporogenesis in Pterocladiella capillacea(Rhodophyta)[J].Journal of Phycology,2012,48(3):710-715.
[28]Koornneef A,Verhage A,Leon-Reyes A,et al.Towards a reporter system to identify regulators of cross-talk between salicylate and jasmonate signaling pathways in Arabidopsis[J].Plant Signaling&Behavior,2008,3(8):543-546.
[29]Wang F,Wang C,Zou T,et al.Comparative transcriptional profiling of Gracilariopsis lemaneiformisinresponse to salicylic acidand methyl jasmonate-mediated heat resistance[J].Plos One,2017,12(5):e0176531.