文心兰不育花粉粒形态观察及相关基因克隆与表达分析
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摘要
该研究以文心兰品种‘柠檬绿’(雄性不育)和‘巧克力’(可育)为试验材料,对花粉团和花粉粒的形态进行观察,并在转录组数据分析的基础上,利用RT-PCR技术从‘柠檬绿’中克隆了1个MYB转录因子基因(OnMYB106),用qRT-PCR技术分析该基因的相对表达量,以初步鉴定‘柠檬绿’花粉败育特征,揭示花粉败育与OnMYB106基因的调控关系。结果显示:(1)‘柠檬绿’花粉团药室明显变扁,外壁凹陷、皱缩。(2)‘柠檬绿’花粉细胞内含物缺失,细胞质空泡化严重,花粉粒形态异常,花粉壁发育不连续,有许多孔洞(并非萌发孔)。(3)成功克隆获得‘柠檬绿’MYB106的基因序列,命名为OnMYB106,其开放阅读框为819bp,编码272个氨基酸;其基因组序列与cDNA比对显示,OnMYB106基因含有3个外显子和2个内含子。(4)生物信息学分析表明,OnMYB106属于SANT超家族,具有2个连续的MYB DNA-binding结构域,是一个典型的R2R3-MYB转录因子;进化树分析表明,OnMYB106与高粱、水稻和二穗短柄草的MYB106蛋白序列的一致性最高,进化距离最近。(5)qRT-PCR分析显示,OnMYB106在‘柠檬绿’花粉团中下调表达;该基因在‘柠檬绿’不同组织器官中均有表达,但在花中表达量最高,假鳞茎中表达量最低;在不同花期中,花苞期表达量最高,盛开期只有微量表达。研究认为,‘柠檬绿’花粉壁发育异常可能导致其花粉败育;OnMYB106基因可能在‘柠檬绿’花器官的生长发育中起重要的调控作用,并且该基因可能属于花粉发育"早期"表达基因,主要影响‘柠檬绿’花粉壁的的形成。
In order to investigate the pollen abortion characteristics of Oncidium Honey Angel and reveal the regulation relationship between pollen abortion and OnMYB106 gene,this study used Oncidium Honey Angel(male sterile)and Oncidium Sharry Baby(fertile)as experimental materials to observe the morphology and characteristics of pollen clusters and pollen grains.In addition,based on the transcriptome analysis,a MYB transcription factor gene(OnMYB106)was cloned from Oncidium Honey Angel by RT-PCR technique,and the relative expression of the gene was analyzed by qRT-PCR.The results showed that:(1)the anther chambers of Oncidium Honey Angel was obviously flattened,and the outer walls were sunken and shrunk.(2)The contents of Oncidium Honey Angel pollen cells were missing and the cytoplasm was vacuolated.Its pollen grains were abnormal,the pollen wall was not continuous,and there were many holes(not germination holes).(3)The OnMYB106 gene contains an open reading frame of 819 bp that encodes 272 amino acids.Comparison between the cDNA and genomic DNA sequences showed that OnMYB106 gene contains three exons and two introns.(4)Bioinformatical analysis revealed that OnMYB106 belongs to the SANT superfamily and has two consecutive MYB DNA-binding domains.It is a typical R2 R3-MYB transcription factor.Phylogenetic tree analysis showed that OnMYB106 had the highest consistency with the MYB106 protein sequence of Sorghum bicolor,Oryza sativa and Brachypodium distachyon,and the evolutionary distance was the closest.(5)Quantitative real time PCR results showed that OnMYB106 was down-regulated in the Oncidium Honey Angel pollen cluster.OnMYB106 was expressed in different tissues of Oncidium Honey Angel,with the highest expression in flowers and the lowest expression in pseudobulbs.In the flowering stage,the expression level of OnMYB106 was the highest in the bud peroid,and only a small amount in the blooming period.This study showed that the abnormal pollen wall development of Oncidium Honey Angel may lead to pollen abortion.The OnMYB106 gene may play an important regulatory role in the growth and development of floral organs,and this gene may belong to the"early"expression genes of pollen development,which mainly affects the formation of pollen wall of Oncidium Honey Angel.
In order to investigate the pollen abortion characteristics of Oncidium Honey Angel and reveal the regulation relationship between pollen abortion and OnMYB106 gene,this study used Oncidium Honey Angel(male sterile)and Oncidium Sharry Baby(fertile)as experimental materials to observe the morphology and characteristics of pollen clusters and pollen grains.In addition,based on the transcriptome analysis,a MYB transcription factor gene(OnMYB106)was cloned from Oncidium Honey Angel by RT-PCR technique,and the relative expression of the gene was analyzed by qRT-PCR.The results showed that:(1)the anther chambers of Oncidium Honey Angel was obviously flattened,and the outer walls were sunken and shrunk.(2)The contents of Oncidium Honey Angel pollen cells were missing and the cytoplasm was vacuolated.Its pollen grains were abnormal,the pollen wall was not continuous,and there were many holes(not germination holes).(3)The OnMYB106 gene contains an open reading frame of 819 bp that encodes 272 amino acids.Comparison between the cDNA and genomic DNA sequences showed that OnMYB106 gene contains three exons and two introns.(4)Bioinformatical analysis revealed that OnMYB106 belongs to the SANT superfamily and has two consecutive MYB DNA-binding domains.It is a typical R2 R3-MYB transcription factor.Phylogenetic tree analysis showed that OnMYB106 had the highest consistency with the MYB106 protein sequence of Sorghum bicolor,Oryza sativa and Brachypodium distachyon,and the evolutionary distance was the closest.(5)Quantitative real time PCR results showed that OnMYB106 was down-regulated in the Oncidium Honey Angel pollen cluster.OnMYB106 was expressed in different tissues of Oncidium Honey Angel,with the highest expression in flowers and the lowest expression in pseudobulbs.In the flowering stage,the expression level of OnMYB106 was the highest in the bud peroid,and only a small amount in the blooming period.This study showed that the abnormal pollen wall development of Oncidium Honey Angel may lead to pollen abortion.The OnMYB106 gene may play an important regulatory role in the growth and development of floral organs,and this gene may belong to the"early"expression genes of pollen development,which mainly affects the formation of pollen wall of Oncidium Honey Angel.
引文
[1] KAUL M L.Male Sterility in Higher Plants[M].New York:Springer Verlag,1988:3-6.
[2]刘忠松,官春云,陈社员.植物雄性不育机理的研究及应用[M].北京:中国农业出版社,2001:10.
[3]朱骏.转录因子MYB103调控花药发育分子机理以及绒毡层发育与功能转录调控途径的研究[D].上海:上海交通大学,2010.
[4]赵会芳,巩振辉,赵利民,等.大白菜胞质雄性不育系RC7及其保持系的细胞学和蕾期基因表达差异分析[J].西北植物学报,2013,33(7):1 317-1 324.ZHAO H F,GONG Z H,ZHAO L M,et al.Differential expression analysis in flower buds of RC7CMS and its maintainer lines of Brassica campestris L[J].Acta Botanica BorealiOccidentalia Sinica,2013,33(7):1 317-1 324.
[5]吴智明.辣椒花蕾内源激素与基因表达差异分析及细胞质雄性不育基因分子标记的研究[D].广州:华南农业大学,2008.
[6]谢潮添,魏冬梅,田惠桥.高等植物雄性不育的细胞生物学研究进展[J].植物生理与分子生物学学报,2006,32(1):17-23.XIE C T,WEI D M,TIAN H Q.Advances in cell biological researches on male sterility of higher plants[J].Journal of Plant Physiology and Molecular Biology,2006,32(1):17-23.
[7] LASER K D,LERSTEN N R.Anatomy and cytology of microsporogenesis in cytoplasmic male sterile angiosperms[J].Botanical Review,1972,38(3):425-454.
[8]邓志刚,金樑,李晶,等.MYB类转录因子对花药和花粉发育的调控途径[J].西北植物学报,2013,33(4):850-856.DENG Z G,JIN L,LI J,et al.Regulatory pathways of MYB transcription factors on anther and pollen development[J].Acta Botanica Boreali-Occidentalia Sinica,2013,33(4):850-856.
[9]钱景华,周步进,孔祥军,等.红麻MYB21基因的克隆、表达及载体构建[J].生物技术通报,2016,32(11):170-179.QIAN J H,ZHOU B J,KONG X J,et al.Cloning,expression analysis and vector construction of MYB21gene in Kenaf[J].Biotechnology Bulletin,2016,32(11):170-179.
[10] ZHANG Z B,ZHU J,GAO J F,et al.Transcription factor AtMYB103is required for anther development by regulating tapetum development,callose dissolution and exine formation in Arabidopsis[J].Plant Journal,2010,52(3):528-538.
[11] ZHANG S,FANG Z J,ZHU J,et al.OsMYB103is required for rice anther development by regulating tapetum development and exine formation[J].Science Bulletin,2010,55(29):3 288-3 297.
[12] MANDAOKAR A,BROWSE J.MYB108acts together with MYB24to regulate jasmonate-mediated stamen maturation in Arabidopsis[J].Plant Physiology,2009,149(2):851-862.
[13]马智明.白菜miR158的表达及其在花粉发育中的功能鉴定[D].杭州:浙江大学,2016.
[14] YEUNG E C,OINAM G S,YEUNG S S,et al.Anther,pollen and tapetum development in safflower,Carthamus tinctorius L[J].Sexual Plant Reproduction,2011,24(4):307-317.
[15]黄鹂.白菜三种雄性不育系与保持系花蕾转录组差异分析及三个花粉发育相关基因功能鉴定[D].杭州:浙江大学,2007.
[16]刘金龙.白菜花粉壁发育相关基因的表达分析与功能验证[D].杭州:浙江大学,2013.
[17] DRAKAKAKI G,ZABOTINA O,DELGADO I,et al.Arabidopsis reversibly glycosylated polypeptides 1and 2are essential for pollen development[J].Plant Physiology,2006,142(4):1 480-1 492.
[18] MOON S,KIM S R,ZHAO G,et al.Rice glycosyltransferase1encodes a glycosyltransferase essential for pollen wall formation[J].Plant Physiology,2013,161(2):663-675.
[19]唐雯.陆地棉隐性核不育系花器发育的形态解剖特征及生理生化特性研究[D].成都:四川农业大学,2008.
[20]陈利维,王春台,余光辉,等.一个水稻MYB基因的功能初步分析[J].华中农业大学学报,2014,33(4):7-12.
[21] STRACKE R,WERBER M,WEISSHAAR B.The R2R3-MYB gene family in Arabidopsis thaliana[J].Current Opinion in Plant Biology,2001,4(5):447-456.
[22] DUBOS C,STRACKE R,GROTEWOLD E,et al.MYB transcription factors in Arabidopsis[J].Trends in Plant Science,2010,15(10):573-581.
[23]WILSON Z A,ZHANG D B.From Arabidopsis to rice:pathways in pollen development[J].Journal of Experimental Botany,2009,60(5):1 479-1 492.
[24] MASCARENHAS J P.Gene activity during pollen development[J].Annu.Rev.Plant Physiol.Plant Mol.Biol,1990,41(1):317-338.
[2]刘忠松,官春云,陈社员.植物雄性不育机理的研究及应用[M].北京:中国农业出版社,2001:10.
[3]朱骏.转录因子MYB103调控花药发育分子机理以及绒毡层发育与功能转录调控途径的研究[D].上海:上海交通大学,2010.
[4]赵会芳,巩振辉,赵利民,等.大白菜胞质雄性不育系RC7及其保持系的细胞学和蕾期基因表达差异分析[J].西北植物学报,2013,33(7):1 317-1 324.ZHAO H F,GONG Z H,ZHAO L M,et al.Differential expression analysis in flower buds of RC7CMS and its maintainer lines of Brassica campestris L[J].Acta Botanica BorealiOccidentalia Sinica,2013,33(7):1 317-1 324.
[5]吴智明.辣椒花蕾内源激素与基因表达差异分析及细胞质雄性不育基因分子标记的研究[D].广州:华南农业大学,2008.
[6]谢潮添,魏冬梅,田惠桥.高等植物雄性不育的细胞生物学研究进展[J].植物生理与分子生物学学报,2006,32(1):17-23.XIE C T,WEI D M,TIAN H Q.Advances in cell biological researches on male sterility of higher plants[J].Journal of Plant Physiology and Molecular Biology,2006,32(1):17-23.
[7] LASER K D,LERSTEN N R.Anatomy and cytology of microsporogenesis in cytoplasmic male sterile angiosperms[J].Botanical Review,1972,38(3):425-454.
[8]邓志刚,金樑,李晶,等.MYB类转录因子对花药和花粉发育的调控途径[J].西北植物学报,2013,33(4):850-856.DENG Z G,JIN L,LI J,et al.Regulatory pathways of MYB transcription factors on anther and pollen development[J].Acta Botanica Boreali-Occidentalia Sinica,2013,33(4):850-856.
[9]钱景华,周步进,孔祥军,等.红麻MYB21基因的克隆、表达及载体构建[J].生物技术通报,2016,32(11):170-179.QIAN J H,ZHOU B J,KONG X J,et al.Cloning,expression analysis and vector construction of MYB21gene in Kenaf[J].Biotechnology Bulletin,2016,32(11):170-179.
[10] ZHANG Z B,ZHU J,GAO J F,et al.Transcription factor AtMYB103is required for anther development by regulating tapetum development,callose dissolution and exine formation in Arabidopsis[J].Plant Journal,2010,52(3):528-538.
[11] ZHANG S,FANG Z J,ZHU J,et al.OsMYB103is required for rice anther development by regulating tapetum development and exine formation[J].Science Bulletin,2010,55(29):3 288-3 297.
[12] MANDAOKAR A,BROWSE J.MYB108acts together with MYB24to regulate jasmonate-mediated stamen maturation in Arabidopsis[J].Plant Physiology,2009,149(2):851-862.
[13]马智明.白菜miR158的表达及其在花粉发育中的功能鉴定[D].杭州:浙江大学,2016.
[14] YEUNG E C,OINAM G S,YEUNG S S,et al.Anther,pollen and tapetum development in safflower,Carthamus tinctorius L[J].Sexual Plant Reproduction,2011,24(4):307-317.
[15]黄鹂.白菜三种雄性不育系与保持系花蕾转录组差异分析及三个花粉发育相关基因功能鉴定[D].杭州:浙江大学,2007.
[16]刘金龙.白菜花粉壁发育相关基因的表达分析与功能验证[D].杭州:浙江大学,2013.
[17] DRAKAKAKI G,ZABOTINA O,DELGADO I,et al.Arabidopsis reversibly glycosylated polypeptides 1and 2are essential for pollen development[J].Plant Physiology,2006,142(4):1 480-1 492.
[18] MOON S,KIM S R,ZHAO G,et al.Rice glycosyltransferase1encodes a glycosyltransferase essential for pollen wall formation[J].Plant Physiology,2013,161(2):663-675.
[19]唐雯.陆地棉隐性核不育系花器发育的形态解剖特征及生理生化特性研究[D].成都:四川农业大学,2008.
[20]陈利维,王春台,余光辉,等.一个水稻MYB基因的功能初步分析[J].华中农业大学学报,2014,33(4):7-12.
[21] STRACKE R,WERBER M,WEISSHAAR B.The R2R3-MYB gene family in Arabidopsis thaliana[J].Current Opinion in Plant Biology,2001,4(5):447-456.
[22] DUBOS C,STRACKE R,GROTEWOLD E,et al.MYB transcription factors in Arabidopsis[J].Trends in Plant Science,2010,15(10):573-581.
[23]WILSON Z A,ZHANG D B.From Arabidopsis to rice:pathways in pollen development[J].Journal of Experimental Botany,2009,60(5):1 479-1 492.
[24] MASCARENHAS J P.Gene activity during pollen development[J].Annu.Rev.Plant Physiol.Plant Mol.Biol,1990,41(1):317-338.