西瓜(Citrullus lanatus)不同雄花发育阶段的转录组分析

doi:10.1016/S2095-3119(21)63615-8

摘要/Abstract

摘要：

西瓜(Citrullus lanatus (Thunb.)Matsum. & Nakai)是世界范围内种植的一种重要的瓜类作物。西瓜果实品质、育性、结实率与雄花发育密切相关。本研究通过细胞学观察，对西瓜新品种‘新特大郑抗9号’雄花发育的不同阶段进行了区分，并进行了转录组测序分析。对花药进行醋酸洋红染色，并测定未开放雄花的纵横径。对花药不同发育时期的细胞学观察表明，花药从四分体生长到成熟期，其纵横径逐渐增大，雄花的花蕾长度在发育过程中也发生了显著的变化。对花药发育的四分体期(A组)、单核期(B组)、双核期(C组)和成熟期(D组)四个发育时期进行转录组测序分析。结果表明，四个阶段总共发现了16288个差异表达基因(DEGs)，随着发育阶段的延伸，各比较组间的DEGs数量逐渐增加，6个比较组(A-VS-B、A-VS-C、A-VS-D、B-VS-C、B-VS-D和C-VS-D)的DEGs分别为2014、3259、4628、1490、3495和1132个。GO和KEGG富集分析表明，DEGs主要富集于细胞组分、淀粉和蔗糖代谢、苯丙类生物合成和戊糖合成等途径。最后，我们在6个比较组中共筛选了59个DEGs，有趣的是，我们发现一个花粉特异表达蛋白(Cla001608)显著下调(log2Fold Change值高达17.32)，这表明它可能在雄花发育中起重要作用。本研究为了解西瓜雄性花发育阶段的分子基础提供了依据，并有助于优势杂交育种。

Abstract: Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is an important cucurbit crop grown worldwide. Watermelon fruit quality, fertility, and seed-setting rate are closely related to male flower development. In this study, the different developmental stages of flower buds of the watermelon cultivar ‘Xinteda Zhengkang 9’ were distinguished by cytological observation, and transcriptome sequencing analysis was performed subsequently. Acetocarmine staining of anthers was performed and the longitudinal and transverse diameters of the unopened male flower buds were measured. Cytological observations of anthers at different developmental stages showed that the anther grew from the tetrad to the mature stage, and the longitudinal and transverse diameters of the flower buds increased. The length of the male flower buds also changed significantly during development. Transcriptome sequencing analysis at four periods, the tetrad (A group), mononuclear (B group), dikaryophase (C group), and mature stages (D group). A total of 16 288 differentially expressed genes (DEGs) were detected in the four stages, with the prolongation of developmental stages, the number of DEGs increased gradually in the comparison groups, there was 2 014, 3 259, 4 628, 1 490, 3 495 and 1 132 DEGs revealed in six comparison groups (A-vs.-B, A-vs.-C, A-vs-D, B-vs.-C, B-vs.-D, and C-vs.-D), respectively. Gene Ontology (GO) and KEGG enrichment analysis showed that the DEGs were mainly enriched in cellular component and starch and sucrose metabolism, phenylpropanoid biosynthesis and pentose sugar, etc. Finally, we completely screened 59 DEGs in the six comparison groups, interestingly, we found one pollen-specific protein (Cla001608) that was significantly down-regulated (the value of log₂Fold Change up to 17.32), which indicated that it may play an important role in the development of male flowers. This work provides insight into the molecular basis of the developmental stages of male flowers in watermelon and may aid in dominant cross breeding.

Key words: watermelon , male flowers development , cytological observation , transcriptome sequencing

. 西瓜(Citrullus lanatus)不同雄花发育阶段的转录组分析 [J]. Journal of Integrative Agriculture, 2022, 21(2): 407-421.

ZHU Ying-chun, YUAN Gao-peng, JIA Sheng-feng, AN Guo-lin, LI Wei-hua, SUN De-xi, LIU Jun-pu. Transcriptomic profiling of watermelon (Citrullus lanatus) provides insights into male flowers development[J]. Journal of Integrative Agriculture, 2022, 21(2): 407-421.

参考文献

Anders S, Huber W. 2010. Differential expression analysis for sequence count data. Genome Biology, 11, R106.
Ashburner M, Ball C A, Blake J A, Botstein D, Butler H. 2000. Gene Ontology: Tool for the unification of biology. Nature Genetics, 25, 25.
Baltz R, Domon C, Pillay D T, Steinmetz A. 1992a. Characterization of a pollen-speciﬁc cDNA from sunﬂower encoding a zinc ﬁnger protein. Plant Journal, 2, 713–721.
Baltz R, Evrard J L, Domon C, Steinmetz A. 1992b. A LIM motif is present in a pollen-specific protein. The Plant Cell, 4, 1465–1466.
Bedinger P. 1992. The remarkable biology of pollen. The Plant Cell, 4, 879–887.
Coen E S, Carpenter R. 2001. Pollen development and its stages in rice (Oryza sativa L.). Chinese Journal of Rice Science, 15, 21–28. (in Chinese)
Collins J K, Wu G, Perkins-Veazie P, Spears K, Claypool P L. 2007. Watermelon consumption increases plasma arginine concentrations in adults. Nutrition, 23, 261–266.
Dai D Y, Yuan L W, Sheng Y Y, Zheng Q. 2018. Transcriptome sequencing of stamen in muskmelon male sterile lines at different developmental stages. Biotechnology Bulletin, 34, 93–100. (in Chinese)
Feng D D. 2008. Fine mapping of Arabidopsis thaliana gene controlling microspores development and cloning of male sterility gene promoters from Arabidopsis. MSc thesis, Shanghai Normal University, China. (in Chinese)
Feng J H, Lu Y G, Liu X D, Xu X X. 2001. Pollen development and its stages in rice (Oryza sativa L.). Chinese Journal of Rice Science, 15, 22–29. (in Chinese)
Finn R D, Jody C, Eddy S R. 2011. HMMER web server: Interactive sequence similarity searching. Nucleic Acids Research, 39, 29–37.
Goldberg R B, Beals T P, Sanders P M. 1993. Anther development: Basic principles and practical applications. The Plant Cell, 5, 1217–1229.
Guo S G, Zhang J G, Sun H, Salse J, Lucas W J, Zhang H, Zheng Y, Mao L Y, Ren Y, Wang Z W, Min J M, Guo X S, Murat F, Ham B K, Zhang Z L, Gao S, Huang M Y, Xu Y M, Zhong S L, Bombarely A, et al. 2013. The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions. Nature Genetics, 45, 51–82.
Higginson T, Li S F, Parish R W. 2003. AtMYB103 regulates tapetum and trichome development in Arabidopsis thaliana. Plant Journal, 35, 177–192.
Ji G, Zhang J, Gong G, Shi J, Zhang H. 2015. Inheritance of sex forms in watermelon (Citrullus lanatus). Scientia Horticultural, 193, 367–373.
Jiang X T, Lin D P. 2007. Discovery of watermelon gynoecious gene gy. Acta Horticulturae Sinica, 34, 141–142. (in Chinese)
Jung K H, Han M J, Lee Y S, Kim Y W, Hwang I. 2005. Rice Undeveloped Tapetum1 is a major regulator of early tapetum development. The Plant Cell, 17, 2705–2722.
Kanehisa M, Goto S. 2000. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Research, 28, 27–30.
Khan M M R, Isshiki S. 2011. Development of a cytoplasmic male-sterile line of eggplant (Solanum melongena L.) with the cytoplasm of Solanum anguivi. Plant Breeding, 130, 256–260.
Koltunow A M, Truettner J, Cox K H, Wallroth M, Goldberg R B. 1990. Different temporal and spatial gene expression patterns occur during anther development. The Plant Cell, 2, 1201–1224.
Kong L, Zhang Y, Ye Z, Liu X, Zhao S. 2007. CPC: Assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Research, 35, 345–349.
Li H, Franck P, Vincent S, Werck-Reichhart D, Diehl P. 2010. Cytochrome P450 family member CYP704B2 catalyzes the ω-hydroxylation of fatty acids and is required for anther cutin biosynthesis and pollen exine formation in rice. The Plant Cell, 22, 173–190.
Liu X, Huang J, Parameswaran S, Ito T, Seubert B. 2009. The SPOROCYTELESS/NOZZLE gene is involved in controlling stamen identity in Arabidopsis. Plant Physiology, 151, 1401–1411.
Liu Z H, Boachon B, Lugan R, Tavares R, Erhardt M, Mutterer J, Demais V, Pateyron S, Brunaud V, Ohnishi T, Pencik A, Achard P, Gong F, Hedden P, Reichhart W D, Renault H. 2015. A conserved cytochrome p450 evolved in seed plants regulates flower maturation. Molecular Plant, 8, 1751–1765.
Manzano S, Martínez C, García J M, Megías Z, Jamilena M. 2014. Involvement of ethylene in sex expression and female flower development in watermelon (Citrullus lanatus). Plant Physiology and Biochemistry, 85, 96–104.
McNeil K J, Smith A G. 2009. A glycine-rich protein that facilitates exine formation during tomato pollen development. Planta, 231, 793–808.
Millar A A, Gubler F. 2005. The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development. The Plant Cell, 17, 705–721.
Mundel C, Baltz R, Eliasson A, Bronner R, Grass N. 2000. A LIM-domain protein from sunflower is localized to the cytoplasm and/or nucleus in a wide variety of tissues and is associated with the phragmoplast in dividing cells. Plant Molecular Biology, 42, 291–302.
Penelope P V, Collins J K, Davis A R, Warren R. 2006. Carotenoid content of 50 watermelon cultivars. Journal of Agricultural and Food Chemistry, 54, 2593.
Pertea M, Pertea G M, Antonescu C M, Chang T C, Mendell J T, Salzberg S L. 2015. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnology, 33, 290–295.
Poole C F, Grimball P C. 1945. Interaction of sex, shape, and weight genes in watermelon. Journal of Agricultural Research, 71, 533–552.
Prothro J, Abdel-Haleem H, Bachlava E. 2013. Quantitative trait loci associated with sex expression in an inter-subspecific watermelon population. Journal of the American Society for Horticulturalence, 138, 125–130.
Rhee S J, Seo M, Jang Y J, Cho S, Lee G P. 2015. Transcriptome profiling of differentially expressed genes in floral buds and flowers of male sterile and fertile lines in watermelon. BMC Genomics, 16, 914.
Rosa J. 1928. The inheritance of flower types in Cucumis and Citrullus. Hilgardia, 3, 233–250.
Scott R J, Spielman M, Dickinson H G. 2004. Stamen structure and function. The Plant Cell, 16, 46–60.
Shan W Y, Ji G J, Zhang J, Gong G Y, Zhao H, Xu Y, Zhang H Y. 2016. Cytomorphological observation on three kinds of sexual type sex differentiation in watermelon. China Vegetables, 1, 49–54. (in Chinese)
Shen S S, Zhang H C, Chen X P, Wu Z H. 2004. Studies on soluble suger, starch, free amino acid and free proline of male sterility in eggplant. Journal of Agricultural University of Hebei, 4, 34–36, 92. (in Chinese)
Singh V K, Mangalam A K, Dwivedi S, Naik S. 1998. Primer premier: Program for design of degenerate primers from a protein sequence. Biotechniques, 24, 318–319. (in Chinese)
Steiner-Lange S, Unte U S, Eckstein L, Yang C, Wilson Z A. 2003. Disruption of Arabidopsis thaliana MYB26 results in male sterility due to non-dehiscent anthers. Plant Journal, 34, 519–528.
Trapnell C, Roberts A, Goff L, Pertea G, Kim D. 2012. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature Protocol, 7, 562.
Ulschan B, Alain T. 2019. Cytochrome P450 enzymes: A driving force of plant diterpene diversity. Phytochemistry, 161, 149–162.
Wang J. 2013. Transcriptomics and metabolomics studies on cytoplasmic male sterility in cotton. MSc thesis, Nanjing Agricultural University, Nanjing. (in Chinese)
Wang W, Tan D Y, Tian Y W, Lin D P. 1998. The cytological studies on watermelon male-sterility material S351–1. Acta Botanica Boreali-Occidentalia Sinica, 3, 361–365. (in Chinese)
Wang X Q, Yuan G Z, Wu Z, He L, Teng N J. 2019. Cloning and expression analysis of LaMYB26 gene in lily. Plant Physiology Journal, 55, 290–300. (in Chinese)
Wilson Z A, Morroll S M, Dawson J, Swarup R, Tighe P J. 2010. The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors. Plant Journal, 28, 27–39.
Xiao G H. 2012. Review of watermelon genes for control of morphological traits and resistance traits. Journal of Hunan Agricultural University (Natural Sciences), 38, 567–573, 564. (in Chinese)
Xing L, Zhang D, Zhao C, Li Y, Ma J. 2016. Shoot bending promotes flower bud formation by miRNA-mediated regulation in apple (Malus domestica Borkh.). Plant Biotechnology Journal, 14, 749–770.
Xing M M, Sun C, Li H L, Hu S L, Lei L. 2018. Integrated analysis of transcriptome and proteome changes related to the Ogura cytoplasmic male sterility in cabbage. PLoS ONE, 13, e0193462.
Xuan D D. 2017. Map-based cloning of two genic male sterility genes in rice (Oryza sativa L.). MSc thesis, Chinese Academy of Agricultural Sciences, Beijing. (in Chinese)
Zhang D, Luo X, Zhu L. 2011. Cytological analysis and genetic control of rice anther development. Journal of Genetics and Genomics, 38, 379–390.
Zeng F Q. 2010. Study on the molecular mechanism and utilization of recessive genic male sterile line S45ab in Brassica napus L. Ph D thesis, Huazhong Agricultural University, Wuhan. (in Chinese)
Zhang S, Fang Z J, Zhu J, Gao J F, Yang Z N. 2010. OsMYB103 is required or rice anther development by regulating tapetum development and exine formation. Chinese Science Bulletin, 55, 3288–3297. (in Chinese)
Zhang Z B, Zhu J, Gao J F, Wang C, Li H. 2007. Transcription factor AtMYB103 is required for anther development by regulating tapetum development, callose dissolution and exine formation in Arabidopsis. Plant Journal, 52, 528–538.
Zhong S, Joung J, Zheng Y, Chen Y, Liu B. 2011. High-throughput illumina strand-specific RNA sequencing library preparation. Cold Spring Harbor Protocols, 8, 5652.
Zhou Z F. 2011. Molecular mechanism of recessive genic male sterility in Brassica napus and functional analysis of related genes. Ph D thesis, Huazhong Agricultural University, Wuhan. (in Chinese)