Transcriptome of Single and Double Tepals of Clematis patens

doi:10.7525/j.issn.1673-5102.2020.01.014

Abstract

Abstract: With the typical Clematis patens ‘Vyvyan Pennell’ which can open either single petal or double petals as the research object, we chose high throughput sequencing to splice and annotate the sequencing data of three different petal types(single petal, semi-double petal and double petal) on the same plant at the same period of C.patens ‘Vyvyan Pennell’, then selected differentially expressed genes to real-time fluorescence quantitative PCR. There were 13.8 GB raw data. A total of 3 075 differentially expressed genes(DEG) were obtained from the three transcriptome libraries by paired comparison, including 649 upregulated DEG and 605 upregulated DEG in the comparison between single and semi-double petals samples(A vs B). The comparison of semi-double flowers with double flowers(B vs C) included 1 046 up-regulated DEG and 721 down-regulated DEG. There were 1 129 upregulation DEG and 859 upregulation DEG in the comparison between single and double flowering(A vs C). There were 134 differentially expressed genes coexisting under three different perianth. By gene functional annotation, 26 genes that might be related to the double petal trait were screened out from the total DEG for cluster analysis, and 10 target genes were randomly selected for fluorescence quantitative PCR verification. PCR results showed that the expression levels of these genes were significantly different in the three floral envelope types of the same plant at the same time of C.patens ‘Vyvyan Pennell’. Finally, the key genes associated with double tepals Clematis conversion were screened out, including MADS-BOX genes PMADS1, AP3, FRUITFULL and FLC. Auxin reactive protein IAA9, auxin input vector, abscisic acid 8' hydroxylase, indoleacetic acid-induced protein ARG7, etc. This study provides basic data and theoretical basis for exploring the molecular mechanism of Clematis double flower.

Key words: Clematis patens, single and double tepals, high throughput sequencing, fluorescence quantitative PCR

CLC Number:

XIONG Yang-Yang, LUO Lin-Li, ZHAO Shi-Hao, ZHOU An-Long, WANG Jin. Transcriptome of Single and Double Tepals of Clematis patens[J]. Bulletin of Botanical Research, 2020, 40(1): 93-105.

References

[1] Zahn L M,Kong H Z,Leebens-Mack J H,et al.The evolution of the SEPALLATA subfamily of MADS-box genes,a preangiosperm origin with multiple duplications throughout angiosperm history[J].Genetics,2005,169(4):2209-2223.
[2] 孙迎坤.山茶花MADS-box家族A类和C类基因克隆及功能分析[D].北京:中国林业科学研究院,2013.Sun Y K.Isolation and function analysis of class A and C genes of MADS-box family from Camellia javonica[D].Beijing:Chinese academy of Forestry,2013.
[3] 钟军珺.榆叶梅花发育相关MADS-box基因克隆和表达模式研究[D].北京:北京林业大学,2016.Zhong J J.Isolation and expression pattern analysis of MADS-box genes related to flower development of flowering plum(Prunus triloba)[D].Beijing:Beijing Forestry University,2016.
[4] 刘志雄,于先泥.日本晚樱同源异型基因PrseAP3的克隆及其在单瓣与重瓣花中的表达分析[J].华中农业大学学报,2012,31(5):578-583.Liu Z X,Yu X N.Cloning and expressing analysis of a floral homeotic gene PrseAP3 from Prunus lannesiana[J].Journal of Huazhong Agricultural University,2012,31(5):578-583.
[5] Yanofsky M F,Ma H,Bowman J L,et al.The protein encoded by the Arabidopsis homeotic gene Agamous resembles transcription factors[J].Nature,1990,346(6279):35-39.
[6] Dubois A,Raymond O,Maene M,et al.Tinkering with the C-function:a molecular frame for the selection of double flowers in cultivated roses[J].PLoS One,2010,5(2):e9288.
[7] Sun Y K,Fan Z Q,Li X L,et al.The APETALA3 and FRUITFUL homologs in Camellia japonica and their roles in double flower domestication[J].Molecular Breeding,2014,33(4):821-834.
[8] 朱高浦,李纪元,范正琪,等.重瓣茶花"红十八学士"MADS-box家族B-function基因序列分析与原核表达[J].热带作物学报,2012,33(6):1077-1083.Zhu G P,Li J Y,Fan Z Q,et al.Sequence analysis and recombinant protein expression of B-function MADS-box gene involved in double flower formation in Camellia japonica ‘Hong Shibaxueshi’[J].Chinese Journal of Tropical Crops,2012,33(6):1077-1083.
[9] 隋娟娟,李晓昕,杨秋燕,等.重瓣百合LiSEP3基因克隆与表达分析[J].南京林业大学学报:自然科学版,2017,41(1):42-48.Sui J J,Li X X,Yang Q Y,et al.Cloning and expression analysis of gene LiSEP3 in double lily[J].Journal of Nanjing Forestry University:Natural Science Edition,2017,41(1):42-48.
[10] 李合生.现代植物生理学:2版[M].北京:高等教育出版社,2006.Li H S.Modern plant physiology:2nd ed[M].Beijing:Higher Education Press,2006.
[11] 何崇单,蔡雨蒙,李萌,等.基于银杏花芽3个分化时期转录组测序的相关基因筛选与表达分析[J].园艺学报,2018,45(8):1479-1490.He C D,Cai Y M,Li M,et al.Screening and expression analysis of related genes based on transcriptome sequencing of ginkgo flower buds at three differentiation stages[J].Acta Horticulturae Sinica,2018,45(8):1479-1490.
[12] 李太强,刘雄芳,万友名,等.基于高通量测序的极小种群野生植物长梗杜鹃转录组分析[J].植物研究,2017,37(6):825-834.Li T Q,Liu X F,Wan Y M,et al.Transcriptome analysis for Rhododendron longipedicellatum(plant species with extremely small populations) based on high throughput sequencing[J].Bulletin of Botanical Research,2017,37(6):825-834.
[13] 孟亚南,张琳,刘召强,等.荷花转录组测序及花青素苷合成相关基因表达分析[J].西南林业大学学报,2018,38(2):61-69.Meng Y N,Zhang L,Liu Z Q,et al.Transcriptome sequencing and expression analysis of anthocyanin synthesis related genes in lotus[J].Journal of Southwest Forestry University,2018,38(2):61-69.
[14] 姚运法,张少平,练冬梅,等.黄秋葵花和果荚转录组测序及类黄酮代谢差异表达分析[J].西北植物学报,2018,38(11):2000-2009.Yao Y F,Zhang S P,Lian D M,et al.Transcriptome sequencing and differential expression analysis of flavonoid metabolism in flowers and fruits of okra[J].Acta Botanica Boreali-Occidentalia Sinica,2018,38(11):2000-2009.
[15] 孙瑞芬,张艳芳,郭树春,等.基于RNA-Seq技术的盐胁迫向日葵转录组信息分析[J].分子植物育种,2015,13(12):2736-2742.Sun R F,Zhang Y F,Guo S C,et al.Analysis on transcriptome of sunflower under salt stress based on RNA-Seq technology[J].Molecular Plant Breeding,2015,13(12):2736-2742.
[16] 董丽君,孟宪红,孔杰,等.基于转录组分析筛选凡纳滨对虾低温胁迫下的差异表达基因[J].中国水产科学,2019,26(1):161-171.Dong L J,Meng X H,Kong J,et al.Screening of differentially expressed genes related to the cold tolerance in Litopenaeus vannamei based on high-throughput transcriptome se-quencing[J].Journal of Fishery Sciences of China,2019,26(1):161-171.
[17] Anders S,Huber W.Differential expression analysis for sequence count data[J].Genome Biology,2010,11(10):R106.
[18] Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT Method[J].Methods,2001,25(4):402-408.
[19] 熊阳阳,王昌命,王锦.基于RNA-seq的铁线莲转录组信息分析[J/OL].分子植物育种,2018:1-9[2019-01-16].http://kns.cnki.net/kcms/detail/46.1068.S.20180906.0944.004.html.Xiong Y Y,Wang C M,Wang J.RNA-seq based transcriptome information analysis of clematis[J/OL].Molecular Plant Breeding,2018:1-9[2019-01-16].http://kns.cnki.net/kcms/detail/46.1068.S.20180906.0944.004.html.
[20] Kanehisa M,Araki M,Goto S,et al.KEGG for linking genomes to life and the environment[J].Nucleic Acids Research,2008,36(S1):480-484.
[21] Trapnell C,Williams B A,Pertea G,et al.Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[J].Nature Biotechnology,2010,28(5):511-515.
[22] 马月萍,戴思兰.高等植物成花分子机理的研究进展[J].分子植物育种,2007,5(S1):21-28.Ma Y P,Dai S L.Research progress in the molecular mechanisms of flowering in higher plants[J].Molecular Plant Breeding,2007,5(S1):21-28.
[23] 袁重要,周岩,李鹏,等.高等观赏植物花器官发育的分子研究进展[J].福建林业科技,2013,40(4):219-224.Yuan Z Y,Zhou Y,Li P,et al.Advances in molecular research of floral organ development of higher ornamental plant[J].Journal of Fujian Forestry Science and Technology,2013,40(4):219-224.