1 College of Agriculture, Guizhou University/Guizhou Branch of National Wheat Improvement Center, Guiyang 550025, China; 2 College of Biological Science and Agriculture, Qiannan Normal University for Nationalities, Duyun 558000, China
Abstract：SBP-box (SQUAMOSA PROMOTER BINDING PROTEIN box) gene family encode a class of plant-specific SBP transcription factors, which are widely distributed in plants and play important functional regulation in plant growth, development and reproduction, including embryogenesis, secondary metabolites biosynthesis, stress responses, etc. Therefore, it has application value in crop improvement. This study used bioinformatics and quantitative real-time PCR (qRT-PCR) to identify and analyze the expression of Sorghum bicolor SbSBP15 gene, and the recombinant SbSBP15 protein was obtained by Escherichia coli prokaryotic expression system. The results showed that the full length of SbSBP15 gene (GenBank No. MT634207) was 1 227 bp, encoding 408 amino acids. Bioinformatics analysis showed that SbSBP15 protein contained a typical SBP domain with a relative molecular weight of 42.19 kD and the isoelectric point was 8.87. Phylogenetic results showed the closest family relationship with maize (Zea mays). Subcellular localization was mainly in the nucleus, and it had strong hydrophilicity. Besides, protein interaction analysis demonstrated that SbSBP15 might interact with protein kinase. qRT-PCR analysis demonstrated that SbSBP15 was mainly expressed in the stalk, and drought and salt stresses could induce SbSBP15 expression. Furthermore, the expression level of SbSBP15 was also enhanced after plant hormone indole acetic acid (IAA) treatment, however, salicylic acid (SA) reduced the expression of SbSBP15. The optimal protein expression of SbSBP15 were in E. coli Rosetta (DE3) strain, inducing temperature at 25 ℃ and isopropyl-β-D-thiogalactopyranoside (IPTG) concentration of 0.8 mmol/L. This study provides a candidate gene for SbSBP15-based molecular genetic study of sorghum, and further provides a basis for studying the biological function of plant SBP15 gene.
[1] 常建忠, 董春林, 张正, 等. 2019. SbSBP5基因参与高粱对干旱胁迫的响应[J]. 植物遗传资源学报, 20(5): 1301-1308. (Chang J Z, Dong C L, Zhang Z, et al.2019. SbSBP5 is involved in response to drought stress in sorghum[J]. Journal of Plant Genetic Resources, 20(5): 1301-1308.) [2] 陈丽. 1997. 植物转录因子的结构与功能[J]. 植物生理学通讯, 33(6): 401-409. (Chen L.1997. Structure and function of plant transcription factors[J]. Plant Physiology Communications, 33(6): 401-409.) [3] 初梦圆, 于延冲. 2019. 影响植物叶片衰老因素的研究进展[J]. 生命科学, 31(2): 178-184. (Chu M Y, Yu Y C.2019. The research progress of factors affecting plant leaf senescence[J]. Chinese Bulletin of Life Sciences, 31(2): 178-184.) [4] 崔梦杰, 王晨, 冷翔鹏, 等. 2018. 葡萄SBP-box转录因子家族的生物信息学分析及其应答激素调控葡萄果实成熟的作用[J]. 南京农业大学学报, 41(3): 429-439. (Cui M J, Wang C, Leng X P, et al.2018. Bioinformatic analysis of the SBP-box transcription factor and function of their regulation modes on berry ripening process by responding to hormones[J]. Journal of Nanjing Agricultural University, 41(3): 429-439.) [5] 代法国, 胡宗利, 陈国平, 等. 2010. 植物特有的SBP-box基因家族的研究进展[J]. 生命科学, 22(2): 155-160. (Dai F G, Hu Z L, Chen G P, et al.2010. Research progress of SBP box gene family specific to plants[J]. Chinese Bulletin of Life Sciences, 22(2): 155-160.) [6] 胡利宗, 王俊生, 刘红占, 等. 2018. 菜豆SBP-box基因家族的分子特征与进化分析[J]. 分子植物育种, 16(14): 4489-4497. (Hu L Z, Wang J S, Liu H Z, et al.2018. Molecular characteristic and evolution analysis of the SBP gene family in common bean[J]. Molecular Plant Breeding, 16(14): 4489-4497.) [7] 季树太, 王佐民, 郭书刚. 2019. 酿酒高粱研究刍议[J]. 酿酒, 46(2): 28-30. (Ji S T, Wang Z M, Guo S G.2019. Discussion on the research of sorghum for liquor making[J]. Liquor Making, 46(2): 28-30.) [8] 刘成, 李晓旭, 苏玉龙, 等. 2015. 普通烟草SBP转录因子家族的全基因组鉴定及其进化和表达分析[J]. 中国烟草科学, 36(4): 1-11. (Liu C, Li X Y, Su Y L, et al.2015. Genome-wide identification, phylogenetic analysis and expression profiling of the SBP transcription factor family in Nicotiana tabacum[J]. Chinese Tobacco Science, 36(4): 1-11.) [9] 吕亮杰, 陈希勇, 胡梦芸, 等. 2018. 小麦SBP基因家族生物信息学分析[J]. 华北农学报, 33(6): 8-16. (Lv L J, Chen X J, Hu M Y, et al.2018. Bioinformatics identification of SBP genes and expression profiles in wheat[J]. Acta Agriculturae Boreali-Sinica, 33(6): 8-16.) [10] 苏顺宗, 吴锋锴, 刘丹, 等. 2013. 一个玉米Pht1家族磷转运蛋白基因克隆和功能分析[J]. 核农学报, 27(7): 885-894. (Su S Z, Wu F K, Liu D, et al.2013. Cloning and characterization of a phosphate transporter gene of Pht1 family in maize[J]. Journal of Nuclear Agricultural Sciences, 27(7): 885-894.) [11] 孙化雨, 杨意宏, 娄永峰, 等. 2017. 毛竹SBP转录因子基因的全基因组鉴定和表达分析[J]. 基因组学与应用生物学, 36(10): 4263-4274. (Sun H Y, Yang Y H, Lou Y F, et al.2017. Genome-wide identification and expression analysis of SBP transcription factor genes in Moso bamboo[J]. Genomics and Applied Biology, 36(10): 4263-4274.) [12] 谢鑫, 蒋君梅, 王勇, 等. 2019. 基于PCR的两步法丝状真菌基因敲除方法[J]. 种子, 38(4): 15-19. (Xie X, Jiang J M, Wang Y, et al.2019. Gene knockout method of filamentous fungi based on two-step PCR amplification[J]. Seed, 38(4): 15-19.) [13] 姚清国. 2017. 运用TMHMM软件对水稻水通道蛋白OsPIP2: 6跨膜结构的分析[J]. 河南农业, 10(29): 59.(Yao Q G. 2017. Analysis of the transmembrane structure of rice aquaporin ospip2: 6 by TMHMM software[J]. Agriculture of Henan, 10(29): 59.) [14] 张晓辉, 魏小春, 李锡香, 等. 2014. 苹果全基因组SBP-box基因家族分析及代表成员的分子克隆[J]. 园艺学报, 41(2): 215-226. (Zhang X H, Wei X C, Li X X, et al.2014. Genome-wide Analysis of SBP-box gene family and molecular cloning of its typical members in Malus×domestica[J]. Acta Horticulturae Sinica, 41(2): 215-226.) [15] 赵雪惠, 王庆杰, 李晨, 等. 2018. 桃ERF转录因子家族生物信息学分析及芽萌发相关基因筛选[J]. 植物学报, 53(5): 612-624. (Zhao X H, Wang Q J, Li C, et al.2018. Genome-wide identification of ethylene responsive factor (ERF) family genes in peach and screening of genes related to germination[J]. Chinese Bulletin of Botany, 53(5): 612-624.) [16] Ali H, Liu Y, Azam S M, et al.2017. Genomic survey, characterization, and expression profile analysis of the SBP genes in pineapple (Ananas comosus L.)[J]. International Journal of Genomics, 14: 1032846. [17] Almagro A J J, Tsirigos K D, Sønderby C K, et al.2019. SignalP 5.0 improves signal peptide predictions using deep neural networks[J]. Nature Biotechnology, 37(4): 420-423. [18] Bušić A, Marđetko N, Kundas S, et al.2018. Bioethanol production from renewable raw materials and its separation and purification: A review[J]. Food Technology and Biotechnology, 56(3): 289-311. [19] Cai C, Guo W, Zhang B.2018. Genome-wide identification and characterization of SPL transcription factor family and their evolution and expression profiling analysis in cotton[J]. Scientific Reports, 8(1): 762. [20] Chen G S, Li J T, Liu Y, et al.2019. Roles of the GA-mediated SPL gene family and miR156 in the floral development of Chinese chestnut (Castanea mollissima)[J]. International Journal of Molecular Sciences, 20(7): 1577. [21] Cheng H T, Hao M Y, Wang W X, et al.2016. Genomic identification, characterization and differential expression analysis of SBP-box gene family in Brassica napus[J]. BMC Plant Biology, 16(1): 196. [22] Chuck G S, Brown P J, Meeley R, et al.2014. Maize SBP-box transcription factors unbranched2 and unbranched3 affect yield traits by regulating the rate of lateral primordia initiation[J]. Proceedings of the National Academy of Sciences of the USA, 111(52): 18775-18780. [23] Guo A Y, Zhu Q H, Gu X C, et al.2008. Genome-wide identification and evolutionary analysis of the plant specific SBP-box transcription factor family[J]. Gene, 418(1-2): 1-8. [24] Han Y C, Gao H Y, Chen H J, et al.2019. The involvement of papaya CpSBP1 in modulating fruit softening and carotenoid accumulation by repressing CpPME1/2 and CpPDS4[J]. Scientia Horticulturae, 256: 108582. [25] Jorgensen S A, Preston J C.2014. Differential SPL gene expression patterns reveal candidate genes underlying flowering time and architectural differences in Mimulus and Arabidopsis[J]. Molecular Phylogenetics and Evolution, 73: 129-139. [26] Kelley L A, Mezulis S, Yates C M, et al.2015. The Phyre2 web portal for protein modeling, prediction and analysis[J]. Nature Protocols, 10(6): 845-858. [27] Lan T, Zheng Y, Su Z, et al.2019. OsSPL10 a SBP-Box Gene, plays a dual role in salt tolerance and trichome formation in rice (Oryza sativa L.)[J]. G3: Genes, Genomes, Genetics, 9(12): 4107-4114. [28] Lei M, Li Z Y, Wang J B, et al.2018. Constitutive expression of Aechmea fasciata SPL14 (AfSPL14) accelerates flowering and changes the plant architecture in Arabidopsis[J]. International Journal of Molecular Sciences, 19(7): 2085. [29] Li J, Li S, Yu M, et al.2014. A cost-effective integrated process to convert sweet sorghum stalks into biofuels[J]. Energy Procedia, 6(1): 2137-2140. [30] Liu M, Sun W, Ma Z, et al.2019. Genome-wide identification of the SPL gene family in tartary buckwheat (Fagopyrum tataricum) and expression analysis during fruit development stages[J]. BMC Plant Biology, 19(1): 299. [31] Luo L, Li W, Miura K, et al.2012. Control of tiller growth of rice by OsSPL14 and strigolactones, which work in two independent pathways[J]. Plant and Cell Physiology, 53(10): 1793-1801. [32] Miura K, Ikeda M, Matsubara A, et al.2010. OsSPL14 promotes panicle branching and higher grain productivity in rice[J]. Nature genetics, 42(6): 545-549. [33] Nuruzzaman M, Sharoni A M, Satoh K, et al.2012. Comprehensive gene expression analysis of the NAC gene family under normal growth conditions, hormone treatment, and drought stress conditions in rice using near-isogenic lines (NILs) generated from crossing Aday Selection (drought tolerant) and IR64[J]. Molecular Genetics and Genomics, 287(5): 389-410. [34] Omidbakhshfard M A, Proost S, Fujikura U, et al.2015. Growth-regulating factors (GRFs): A small transcription factor family with important functions in plant biology[J]. Molecular Plant, 8(7): 998-1010. [35] Ran L, Ding Y, Luo L, et al.2017. Interaction research on an antiviral molecule that targets the coat protein of Southern rice black-streaked dwarf virus[J]. International Journal of Biological Macromolecules, 103: 919-930. [36] Shao Y, Zhou H Z, Wu Y, et al.2019. OsSPL3, an SBP-Domain Protein, regulates crown root development in rice[J]. The Plant Cell, 31(6): 1257-1275. [37] Silva G F F, Silva E M, Azevedo M S, et al.2014. micro RNA 156-targeted SPL/SBP box transcription factors regulate tomato ovary and fruit development[J]. The Plant Journal, 78(4): 604-618. [38] Stone J M, Liang X, Nekl E R, et al.2005. Arabidopsis AtSPL14, a plant specific SBP-domain transcription factor, participates in plant development and sensitivity to fumonisin B1[J]. The Plant Journal, 41(5): 744-754. [39] Tan H W, Song X M, Duan W K, et al.2015. Genome-wide analysis of the SBP-box gene family in Chinese cabbage (Brassica rapa subsp. pekinensis)[J]. Genome, 58(11): 463-477. [40] Wang L, Sun S Y, Jin J Y, et al.2015. Coordinated regulation of vegetative and reproductive branching in rice[J]. Proceedings of the National Academy of Sciences of the USA, 112(50): 15504-15509. [41] Wang P J, Chen D, Zheng Y C, et al.2018. Identification and expression analyses of SBP-box genes reveal their involvement in abiotic stress and hormone response in tea plant (Camellia sinensis)[J]. International Journal of Molecular Sciences, 19(11): 3404. [42] Wang R F, Tavano E C D R, Lammers M, et al.2019. Re-evaluation of transcription factor function in tomato fruit development and ripening with CRISPR/Cas9-mutagenesis[J]. Scientific Reports, 9(1): 1696. [43] Xing X J, Jiang J F, Huang Y Y, et al.2019. The constitutive expression of a Chrysanthemum ERF transcription factor influences flowering time in Arabidopsis thaliana[J]. Molecular Biotechnology, 61(1): 20-31. [44] Zhou Q, Zhang S, Chen F, et al.2018. Genome-wide identification and characterization of the SBP-box gene family in Petunia[J]. BMC Genomics, 19(1): 193.
