甜瓜雄性不育两用系转录组测序与激素含量研究
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摘要
为了从分子及生理生化水平探讨甜瓜雄性不育发生过程中内源激素(生长素、赤霉素、茉莉酸、水杨酸)与花粉败育的相关性,利用雄性不育两用系对甜瓜可育株与不育株2 mm花蕾雄蕊进行转录组测序,筛选内源激素相关差异表达基因,结合实时荧光定量分析以鉴定差异基因表达量,并对雄性不育株与可育株的花蕾内源激素含量进行测定。结果显示:转录组测序共发现334个差异表达基因,对差异表达基因进行GO功能分析显示,共有273个基因归属于生物合成、分子功能和细胞组分三大分支,其中与内源激素相关的差异基因为7个,其中2个差异表达基因为水杨酸甲基转移同源基因,3个为生长素相关表达基因,赤霉素相关表达基因和茉莉酸相关表达基因各1个;qRT-PCR验证表明,与生长素相关的基因和吲哚-3-乙酸合成相关基因在雄性不育株花蕾中的表达量均高于可育株的表达量,而赤霉素、茉莉酸和水杨酸相关表达基因在雄性不育株花蕾中的表达量则是低于可育株中的表达量。激素含量的测定结果显示赤霉素和生长素含量可育株高于不育株,茉莉酸甲酯与水杨酸的含量不育株高于可育株。研究结果表明植物激素类相关基因通过间接方式参与雄性不育发生过程,同时多个激素基因之间存在相互拮抗,代谢途径复杂;茉莉酸甲酯和水杨酸含量过度积累可能与雄性不育的发生相关。
In order to explore the relationship between the occurrence of male sterility in melon(Cucumis melo L.) and the metabolism of hormone(Indole-3-acetic acid IAA,Gibberellin GA,Jasmonic acid methyl esterfrom Ja Me,and Salicylic acid SA)in the transcriptional and physiological and biochemical level,the RNA-seq by the stamens of the fertile and sterile plants of 2 mm buds were conducted in the present study,deferentially expressed genes related to the hormone were screened,the real time fluorescence quantitative analysis in 2 mm diameter stage of microspore development in MS and FS plants were carried out to identify the expression pattern of differential genes,hormone contents were also investigated in floral buds of male sterile and fertile plants. The results showed that a total of 334 differentially expressed genes were found out,273 genes in transcriptome sequencing,GO function analysis showed these genes were involved in the three branches including the biosynthesis,molecular function and cell component. Among them,7 genes related to the hormone including 2 Ja Me genes,3 IAA-related genes,1 GA-related gene and 1 SA-related gene. qRTPCR validation showed that fertile plants' expression of IAA related genes and Indole-3-acetic acid related genes were higher than sterile plants,on the contrary,fertile plants' expression of GA,Ja Me and SA related gene were lower than sterile plants,the hormone content of GA and IAA in male fertility plants were higher than that of sterility plants,while Ja Me and SA content in male sterility were higher than fertility lines.The results supposed that the hormone related gene participate in occurrence of male sterility with indirect way,different hormone related gene interact with each other,and the pathway was complex,the excessive accumulation of Ja Me and SA might relate to male sterility.
In order to explore the relationship between the occurrence of male sterility in melon(Cucumis melo L.) and the metabolism of hormone(Indole-3-acetic acid IAA,Gibberellin GA,Jasmonic acid methyl esterfrom Ja Me,and Salicylic acid SA)in the transcriptional and physiological and biochemical level,the RNA-seq by the stamens of the fertile and sterile plants of 2 mm buds were conducted in the present study,deferentially expressed genes related to the hormone were screened,the real time fluorescence quantitative analysis in 2 mm diameter stage of microspore development in MS and FS plants were carried out to identify the expression pattern of differential genes,hormone contents were also investigated in floral buds of male sterile and fertile plants. The results showed that a total of 334 differentially expressed genes were found out,273 genes in transcriptome sequencing,GO function analysis showed these genes were involved in the three branches including the biosynthesis,molecular function and cell component. Among them,7 genes related to the hormone including 2 Ja Me genes,3 IAA-related genes,1 GA-related gene and 1 SA-related gene. qRTPCR validation showed that fertile plants' expression of IAA related genes and Indole-3-acetic acid related genes were higher than sterile plants,on the contrary,fertile plants' expression of GA,Ja Me and SA related gene were lower than sterile plants,the hormone content of GA and IAA in male fertility plants were higher than that of sterility plants,while Ja Me and SA content in male sterility were higher than fertility lines.The results supposed that the hormone related gene participate in occurrence of male sterility with indirect way,different hormone related gene interact with each other,and the pathway was complex,the excessive accumulation of Ja Me and SA might relate to male sterility.
引文
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[23]王强,张建农,李计红.甜瓜性别分化的显微结构[J].甘肃农业大学学报,2009(6):79-84.
[24]Shemesh-Mayer E,Ben-Michael T,Rotem N,et al.Garlic(Allium sativum L.)fertility:transcriptome and proteome analyses provide insight into flower and pollen development[J].Front Plant Sci,2015(6):271.
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[26]解海岩,蒋培东,王晓玲,等.棉花细胞质雄性不育花药败育过程中内源激素的变化[J].作物学报,2006,32(7):1094-1096.
[27]白薇,田自华,盖连玉,等.甜菜胞质雄性不育系与其保持系激素含量的差异[J].内蒙古农业大学学报:自然科学版,2004,25(3):48-51.
[28]侯凯,陈郡雯,申浩,等.川白芷内源激素的提取纯化和高效液相色谱法同步测定[J].核农学报,2013,27(5):653-657.
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[30]许小勇,张静,孙希禄,等.大白菜CMS7311雄性不育的发生与花蕾内源激素含量变化的关系研究[J].华北农学报,2014,29(6):177-182.
[31]曾爱松,梁毅,严继勇,等.洋葱胞质雄性不育系及其保持系花蕾内源激素含量和脯氨酸含量的动态变化特征[J].华北农学报,2012,27(S1):77-80.
[32]许忠民,张恩慧,程永安,等.甘蓝胞质雄性不育与内源激素含量的关系[J].北方园艺,2015(8):1-5.
[32]Xie D,Feys B,James S,et al.COIl:An Arabidopsis gene required for jasmonate-regulated defense and fertility[J].Science,1998,280:1091-1094.
[34]Stintzi A,Browse J.The Arabidopsis male-sterile mu-tant,opr3,lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis[J].Proceedings of the National Academy of Sciences of the United States of America,2000,19(97):10625-10630.
[35]宋平,夏凯,吴传万,等.雄性不育和可育水稻开颖对茉莉酸甲酯响应的差异[J].植物学报,2001,43(5):480-485.
[2]祁云霞,刘永斌,荣威恒.转录组研究新技术:RNASeq及其应用[J].遗传,2011,33(11):1191-1202.
[3]Grabherr MG,Haas BJ,Yassour M,et al.Full-length transcriptome assembly from RNA-Seq data without a reference genome[J].Nat Biotechnol,2011,29(7):644-652.
[4]Gómez JF,Talle B,Wilson ZA.Anther and pollen development:A conserved developmental pathway[J].J Integr Plant Biol,2015,57(11):876-891.
[5]陶兴林,侯栋,朱惠霞,等.花椰菜温敏雄性不育系GS-19花药败育的细胞学及转录组分析[J].中国农业科学,2017,50(13):2538-2552.
[6]毛伟兵,陈发菊,王长兰,等.楸树雄性不育花芽转录组测序及分析[J].林业科学,2017(57):141-150.
[7]孙超.甘蓝Ogu CMS花药败育特征及转录组分析[D].兰州:甘肃农业大学,2016.
[8]魏利斌,苗红梅,张海洋.芝麻发育转录组分析[J].中国农业科学,2012,45(7):1246-1256.
[9]Chen LT,Liu YG.Male sterility and fertility restoration in crops[J].Annu Rev Plant Biol,2014,65(1):579-606.
[10]刘永明,张玲,邱涛,等.高通量转录组测序技术在植物雄性不育研究中的应用[J].遗传,2016,38(8):677-687.
[11]Tang ZH,Zhang LP,Xu CG,et al.Uncovering small RNAmediated responses to cold stress in a wheat thermosensitive genic male-sterile line by deep sequencing[J].Plant Physiol,2012,159(2):721-738.
[12]Yang P,Han JF,Huang JL.Transcriptome sequencing and de novo analysis of cytoplasmic male sterility and maintenance in JA-CMS cotton[J].PLo S One,2014,9(11):112320.
[13]Yu JH,Zhao YX,Qin YT,et al.Discovery of micro RNAs associated with the S type cytoplasmic male sterility in maize[J].J Integr Agr,2013,12(2):229-238.
[14]Chen CM,Chen GJ,Cao BH,et al,Transcriptional profiling analysis of genic male sterile-fertile Capsicum annuum reveal candidate genes for pollen development and maturation by RNA-Seq technology[J].Plant Cell Tiss Organ Cult(PCTOC),2015,122(2):465-476.
[15]Jeong HJ,Kang JH,Zhao MA,et al.Tomato Male sterile1035 is essential for pollen development and meiosis in anthers[J].J Exp Bot,2014,65(22):6693-6709.
[16]Jiang JX,Lv ML,Liang Y,et al.Identification of novel and conserved mi RNAs involved in pollen development in Brassica campestris ssp.chinensis by high-throughput sequencing and degradome analysis[J].BMC Genomics,2014,15(1):146.
[17]王薇薇,羊杏平,范淑英,等.甜瓜小孢子发育时期与花器形态的关系[J].西北农林科技大学学报:自然科学版,2015,43(4):108-112.
[18]Rhee SJ,Seo M,Jang JY,et al.Transcriptome profiling of differentially expressed genes in floral buds and flowers of male sterile and fertile lines in watermelon[J].BMC Genomics,2015,16:914.
[19]刘红艳,吴坤,杨敏敏,等.芝麻显性细胞核雄性不育系内源激素、可溶性糖和淀粉含量变化[J].中国油料作物学报,2014,36(2):175-180.
[20]关天霞,党占海,张建平,等.亚麻温敏型雄性不育系花蕾发育过程中内源激素的变化[J].中国油料作物学报,2007,29(3):248-253.
[21]Schulze SK,Kanwar R,G lzenleuchteret M,et al.SERE:Single-parameter quality control and sample comparison for RNA-Seq[J].BMC genomics,2012,13(1):524.
[22]Li JJ,Han SH,Ding XL,et al.Comparative transcriptome analysis between the cytoplasmic male sterile line NJCMS1A and its maintainer NJCMS1B in soybean(Glycine max(L.)Merr.)[J].PLo S One,2015,10(5):126771.
[23]王强,张建农,李计红.甜瓜性别分化的显微结构[J].甘肃农业大学学报,2009(6):79-84.
[24]Shemesh-Mayer E,Ben-Michael T,Rotem N,et al.Garlic(Allium sativum L.)fertility:transcriptome and proteome analyses provide insight into flower and pollen development[J].Front Plant Sci,2015(6):271.
[25]王永勤,曹家树,虞慧芳,等.白菜核雄性不育两用系生理生化特性的分析[J].园艺学报,2003,63(2):212-214.
[26]解海岩,蒋培东,王晓玲,等.棉花细胞质雄性不育花药败育过程中内源激素的变化[J].作物学报,2006,32(7):1094-1096.
[27]白薇,田自华,盖连玉,等.甜菜胞质雄性不育系与其保持系激素含量的差异[J].内蒙古农业大学学报:自然科学版,2004,25(3):48-51.
[28]侯凯,陈郡雯,申浩,等.川白芷内源激素的提取纯化和高效液相色谱法同步测定[J].核农学报,2013,27(5):653-657.
[29]杨晓丽.芝麻核雄性不育的超微结构观察、内源激素测定及相关基因的克隆研究[D].南京:南京农业大学,2008.
[30]许小勇,张静,孙希禄,等.大白菜CMS7311雄性不育的发生与花蕾内源激素含量变化的关系研究[J].华北农学报,2014,29(6):177-182.
[31]曾爱松,梁毅,严继勇,等.洋葱胞质雄性不育系及其保持系花蕾内源激素含量和脯氨酸含量的动态变化特征[J].华北农学报,2012,27(S1):77-80.
[32]许忠民,张恩慧,程永安,等.甘蓝胞质雄性不育与内源激素含量的关系[J].北方园艺,2015(8):1-5.
[32]Xie D,Feys B,James S,et al.COIl:An Arabidopsis gene required for jasmonate-regulated defense and fertility[J].Science,1998,280:1091-1094.
[34]Stintzi A,Browse J.The Arabidopsis male-sterile mu-tant,opr3,lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis[J].Proceedings of the National Academy of Sciences of the United States of America,2000,19(97):10625-10630.
[35]宋平,夏凯,吴传万,等.雄性不育和可育水稻开颖对茉莉酸甲酯响应的差异[J].植物学报,2001,43(5):480-485.