DOI: 10.3724/SP.J.1006.2018.00177

Acta Agronomica Sinica (作物学报) 2018/44:2 PP.177-184

Cloning and Expression Analysis of BoSPI Induced by Self-pollination in Brassica oleracea L. var. capitata

Self incompatibility is a complex and comprehensive genetic mechanism formed in the long-term evolution, which prevents inbreeding and promotes heterosis. Mining functional genes involving in self incompatibility has an important significance for the study of self incompatibility in Brassica oleracea L. var. capitata. In this study, we identified a gene named BoSPI which expression was up-regulated and induced by self-pollination based on the stigma transcriptome data in 0-60 min self-pollination and cross-pollination. BoSPI contains an open reading frame (ORF) with the length of 534 bp, encoding a protein of 177 amino acid residues without introns, which contains four conserved EF-hand domains without signal peptide and transmembrane domain, the theory isoelectric point of BoSPI is 4.21. In addition, diverse cis-acting promoter elements involved in fungal elicitor response, metabolic regulation and organ formation were discovered in the upstream 2000 bp of initial codon of BoSPI. BoSPI could be expressed as a 17 kD protein in E. coli BL21 (DE3). The expression level of BoSPI was the highest in stigmas and lower in petals, sepals, leaves and stamens of cabbage after self-pollination. Subcellular localization analysis revealed that BoSPI encoded a protein localized in the cell membrane and cytoplasm. The expression of BoSPI gene was significantly induced by self pollination after 30 minutes. These results suggest that BoSPI is involved in the molecular processes of the stigma response to self-pollen stimulation, which may be a new functional gene related to the self incompatibility of Brassica oleracea L. var. capitata.

Key words:self-pollination,Brassica oleracea L.var.capitata,gene (BoSPI),transcriptome technology

ReleaseDate:2018-03-08 10:13:39

[1] Chapman L A, Goring D R. Pollen-pistil interactions regulating successful fertilization in the Brassicaceae. J Exp Bot, 2010, 61:1987-1999

[2] Vanoosthuyse V, Tichtinsky G, Dumas C, Gaude T, Cock M. Interaction of calmodulin, a sorting nexin and kinase-associated protein phosphatase with the Brassica oleracea S locus receptor kinase. Plant Physiol, 2003, 133:919-929

[3] Kachroo A, Schopfer C R, Nasrallah M E, Nasrallah J B. Alelle-specific receptor-ligand interactions in Brassica self incompatibility. Science, 2001, 293:1824-1826

[4] Takayama S, Shimosato H, Shiba H, Funato M, Iwano M, Che F S, Watanabe M, Iwano M, Isogai A. Direct ligand-receptor complex interaction controls Brassica self-incompatibility. Nature, 2001, 413:534-538

[5] Newbigin E, Vierstra R D. Plant reproduction:sex and self-denial. Nature, 2003, 423:229-230

[6] 朱利泉, 周燕. 甘蓝自交不亲和性信号传导元件与传导过程. 作物学报, 2015, 41:1-14 Zhu L Q, Zhou Y. Protein elements and signal transduction process of self-incompatibility in Brassica oleracea. Acta Agron Sin, 2015, 41:1-14(in Chinese with English abstract)

[7] Nasrallah J B, Nasrallah M E. Robust self-incompatibility in the absence of a functional ARC1 gene in Arabidopsis thaliana. Plant Cell, 2014, 26:3838-3841

[8] Kitashiba H, Liu P, Nishio T, Nasrallah J B, Nasrallah M E. Functional test of Brassica self-incompatibility modifiers in Arabidopsis thaliana. Proc Natl Acad Sci USA, 2011, 108:18173-18178

[9] 贾新平, 叶晓青, 梁丽建, 邓衍明, 孙晓波, 佘建明. 基于高通量测序的海滨雀稗转录组学研究. 草业学报, 2014, 23:242-252 Jia X P, Ye X Q, Liang L J, Deng Y M, Sun X B, She J M. Transcriptome characteristics of Paspalum vaginatum analyzed with illumina sequencing technology. Acta Pratac Sin, 2014, 23:242-252(in Chinese with English abstract)

[10] Mortazavil A, Williams B A, McCue1 K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods, 2008, 5:621-628

[11] Skelton N J, Kördel J, Akke M, Chazin W. Signal transduction versus buffering activity in Ca2+-binding proteins. Nat Struct Mol Biol, 1994, 1:239-245

[12] Ikura M. Calcium binding and conformational response in EF-hand proteins. Trends Biochem Sci, 1996, 21:14-17

[13] Kretsinger R H. EF-hands embrace. Nat Struct Mol Biol, 1997, 4:514-516

[14] Chen C, Sun X L, Duanmu H Z, Zhu D, Yu Y, Cao L, Liu A, Bowei J, Xiao J L, Zhu Y Z. GsCML27, a gene encoding a calcium-binding EF-hand protein from Glycine soja, plays differential roles in plant responses to bicarbonate, salt and osmotic stresses. PLoS One, 2015, 10:e0141888

[15] 于晓俊, 曹绍玉, 董玉梅, 毕保良, 张应华, 许俊强. 钙结合蛋白对花粉生长发育调控研究进展. 西北植物学报, 2016, 36:2121-2127 Yu X J, Cao S Y, Dong Y M, Bi B L, Zhang Y H, Xu J Q. Research progress of calcium binding proteins in pollen growth and development. Acta Bot Boreali-Occident Sin, 2016, 36:2121-2127(in Chinese with English abstract)

[16] Zonia L, Munnik T. Uncovering hidden treasures in pollen tube growth mechanics. Trends Plant Sci, 2009, 14:318-327

[17] Iwano M, Shiba H, Miwa T, Che F S, Takayama S, Nagai T, Miyawaki A, Lsogai A. Ca2+dynamics in a pollen grain and papilla cell during pollination of Arabidopsis. Plant Physiol, 2004, 136:3562-3571

[18] Lazzaro M D, Cardenas L, Bhatt A P, Justus C D, Phillips M S, Holdaway-Clarke T L, Hepler P K. Calcium gradients in conifer pollen tubes, dynamic properties differ from those seen in angiosperms. J Exp Bot, 2005, 56:2619-2628

[19] Guan Y F, Gu J Z, Li H, Li H, Yang Z B.Signaling in pollen tube growth:crosstalk, feedback and missing links. Mol Plant, 2005, 6:1053-1064

[20] Michard E, Lima P T, Borges F, Silva A C, Portes M T, Carvalho J E, Gilliham M, Liu L H, Obermeyer G, Feijó J A. Glutamate receptor-like genes form Ca2+ channels in pollen tubes and are regulated by pistil D-serine. Science, 2011, 332:434-437

[21] Konrad K R, Wudick M M, Feijó J A. Calcium regulation of tip growth:new genes for old mechanisms. Curr Opin Plant Biol, 2011, 14:721-730

[22] Dearnaley J D W, Levina N N, Lew R R, Heath B, Goring D R. Interrelationships between cytoplasmic Ca2+peaks, pollen hydration and plasma membrane conductances during compatible and incompatible pollinations of Brassica napus papillae. Plant Cell Physiol, 1997, 38:985-999

[23] Elleman C J, Dickinson H G. Commonalities between pollen/stigma and host/pathogen interactions:calcium accumulation during stigmatic penetration by Brassica oleracea pollen tubes. Sex Plant Reprod, 1999, 12:194-202

[24] Goring D R. The search for components of the self-incompatibility signalling pathway (s) in Brassica napus. Ann Bot, 2000, 85(suppl-1):171-179

[25] Franklin-Tong V E, Hackett G, Hepler P K. Ratio-imaging of Ca2+ in the self-incompatibility response in pollen tubes of Papaver rhoeas. Plant J, 1997, 12:1375-1386

[26] Wheeler M J, De Graaf B H J, Hadjiosif N, Perry R M, Poulter N S, Osman K, Vatovec S, Harper A, Franklin F C H, Franklin-Tong V E. Identification of the pollen self-incompatibility determinant in Papaver rhoeas. Nature, 2009, 459:992-995

[27] Iwano M, Ito K, Fujii S, Kakita M, Asano-Shimosato H, Lgarashi M, Kaothien-Nakayama P, Entani T, Kanatani A, Takshisa M, Tanaka M, Komatsu K, Shiba H, Nagai T, Miyawaki A, Isogai A, Takayama S T. Calcium signalling mediates self-incompatibility response in the Brassicaceae. Nat Plants, 2015, 1:15128