Abstract In the process of plant regeneration mediated by developmental regulators, overexpression of developmental regulators could lead to pleiotropic effects such as developmental malformation and pollen abortion. Therefore, finding a suitable promoter to control the suitable expression of developmental regulators in plants may be one of the methods to obtain normal regenerated plants. LEAFY COTYLEDON2 (LEC2), a specific B3 transcription factor in plant embryogenesis, plays a crucial regulatory role in seed maturation. In the study, the results of semi-quantitative PCR showed that GbLEC2 was not or weakly expressed in roots, stems, leaves or embryogenic callus in Gossypium barbadense, but significantly highly expressed in globular embryo, torpedo embryo and cotyledon embryo. Bioinformatics analysis displayed that there were 2 seed-specific cis-elements Skn-1 element and RY repeat element in GbLEC2 promoter sequence. To further identify the transcriptional characters of GbLEC2 promoter, 4 different GbLEC2 promoter fragments including ProGbLEC2-1 (2000 bp), ProGbLEC2-2 (1500 bp), ProGbLEC2-3 (1000 bp) and ProGbLEC2-4 (500 bp) were constructed and then transformed into the different cotton tissues and cells by Agrobacterium-mediated transient transformation method. And the GUS activity assay demonstrated that ProGbLEC2-3 (1000 bp) and ProGbLEC2-4 (500 bp) promoter fragments displayed a specific transcriptional activity in somatic embryogenesis, which preliminarily met the Lowe's requirements for the promoters to drive suitable expression of developmental regulators in plant. Therefore, the GbLEC2 promoter fragment cloned in the study is expected to be a new candidate promoter to precisely regulate the expression of developmental regulators, and thus be applied to a novel molecular breeding technolody platform mediated by developmental regulators-promoting plant regenesis in G. barbadense.
[1] 杨瑞思. 2020. 海岛棉新海 16 体胚发生体系的优化及 Gb-GAI4 基因在海岛棉体胚发生过程中的功能研究[D]. 硕士学位论文, 新疆农业大学, 导师: 张霞, pp. 14-18 (Yang R S. 2020. Optimization of the somatic embryo-genesis system in Gossypium barbadense Xinhai 16 and of functional identification of GbGAI4 gene in G. bar-badense somatic embryogenesis[D]. Thesis for M. S., Xinjiang Agriculture University, Supervisor: Zhang X, pp. 14-18. ) [2] Aoyagi T, Kobayashi M, Kozaki A. 2018. Design of a seed-specific chimeric promoter with a modified expression profile to improve seed oil content[J]. International Jour-nal of Molecular Sciences, 19(6): 1667-1668. [3] Bäumlein H, Nagy I, Villarroel R, et al. 1992. Cis -analysis of a seed protein gene promoter: The conservative RY re-peat CATGCATG within the legumin box is essential for tissue -specific expression of a legumin gene[J]. The Plant Journal, 2(2): 233-239. [4] Boutilier K, Offringa R, Sharma V K, et al. 2002. Ectopic ex-pression of BABY BOOM triggers a conversion from vegetative to embryonic growth[J]. The Plant Cell, 14(8): 1737-1749. [5] Braybrook S A, Stone S L, Park S, et al. 2006. Genes directly regulated by LEAFY COTYLEDON2 provide insight into the control of embryo maturation and somatic embryo-genesis[J]. Proceedings of the National Academy of Sci-ences of the USA, 103(9): 3468-3473. [6] Guo F, Liu C, Xia H, et al. 2013. Induced expression of AtLEC1 and AtLEC2 differentially promotes somatic em-bryogenesis in transgenic tobacco plants[J]. PLOS One, 8(8): e71714. [7] Gong P, Wei R, Li Y, et al. 2019. Molecular cloning and functional characterization of a seed-specific VvβVPE gene promoter from Vitis vinifera[J]. Planta, 250(2): 657-665. [8] Harding E W, Tang W, Nichols K W, et al. 2003. Expression and maintenance of embryogenic potential is enhanced through constitutive expression of AGAMOUS-Like 15[J]. Plant Physiology, 133(2): 653-663. [9] Jones T, Lowe K, Hoerster G, et al. 2019. Maize transforma-tion using the morphogenic genes Baby Boom and Wus-chel2[M]. Methods in Molecular Biology (Clifton, N. J. ), 1864: 81-93 [10] Kim H U, Jung S J, Lee K R, et al. 2014. Ectopic overex-pression of castor bean LEAFY COTYLEDON2 (LEC2) in Arabidopsis triggers the expression of genes that en-code regulators of seed maturation and oil body pro-teins in vegetative tissues[J]. FEBS Open Bio, 4: 25-32. [11] Lenhard M, Jürgens G, Laux T. 2002. The WUSCHEL and SHOOTMERISTEMLESS genes fulfil complementary roles in Arabidopsis shoot meristem regulation[J]. Devel-opment, 129(13): 3195-3206 [12] Li H, Li K, Guo Y, et al. 2018. A transient transformation sys-tem for gene characterization in upland cotton (Gossypi-um hirsutum)[J]. Plant Methods, 14(1): 1-11. [13] Lotan T, Ohto M, Yee K M, et al. 1998. Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo develop-ment in vegetative cells[J]. Cell, 93(7): 1195-1205. [14] Lowe K, Wu E, Wang N, et al. 2016. Morphogenic regulators Baby boom and Wuschel improve monocot transforma-tion[J]. The Plant Cell, 28(9): 1998-2015. [15] Lowe K, La Rota M, Hoerster G, et al. 2018. Rapid genotype “independent”Zea mays L. (maize) transformation via direct somatic embryogenesis[J]. In Vitro Cellular & De-velopmental Biology-Plant, 54(3): 240-252. [16] Rikiishi K, Maekawa M. 2014. Seed maturation regulators are related to the control of seed dormancy in wheat (Triti-cum aestivum L. )[J]. PLOS One, 9(9): e107618. [17] Santos -Mendoza M, Dubreucq B, Baud S, et al. 2008. Deci-phering gene regulatory networks that control seed de-velopment and maturation in Arabidopsis[J]. The Plant Journal, 54(4): 608-620. [18] Schmidt E D, Guzzo F, Toonen M A, et al. 1997. A leucine-rich repeat containing receptor-like kinase marks somat-ic plant cells competent to form embryos[J]. Develop-ment, 124(10): 2049-2062. [19] Stone S L, Kwong L W, Yee K M, et al. 2001. LEAFY COTY-LEDON2 encodes a B3 domain transcription factor that induces embryo development[J]. Proceedings of the Na-tional Academy of Sciences of the USA, 98(20): 11806-11811. [20] Suzuki M, Wang H H Y, McCarty D R. 2007. Repression of the LEAFY COTYLEDON 1/B3 regulatory network in plant embryo development by VP1/ABSCISIC ACID IN-SENSITIVE 3-LIKE B3 genes[J]. Plant Physiology, 143(2): 902-911. [21] Thakare D, Tang W, Hill K, et al. 2008. The MADS-domain transcriptional regulator AGAMOUS-LIKE15 promotes somatic embryo development in Arabidopsis and soybean[J]. Plant Physiology, 146(4): 1663-1672. [22] Tvorogova V E, Lutova L A. 2018. Genetic regulation of zy-gotic embryogenesis in angiosperm plants[J]. Russian Journal of Plant Physiology, 65(1): 1-14. [23] Wang H, Guo J, Lambert K N, et al. 2007. Developmental control of Arabidopsis seed oil biosynthesis[J]. Planta, 226(3): 773-783. [24] Washida H, Wu C Y, Suzuki A, et al. 1999. Identification of cis-regulatory elements required for endosperm expres-sion of the rice storage protein glutelin gene GluB-1[J]. Plant Molecular Biology, 40(1): 1-12. [25] Zhang Y, Clemens A, Maximova S N, et al. 2014. The Theo-broma cacao B3 domain transcription factor TcLEC2 plays a duel role in control of embryo development and maturation[J]. BMC Plant Biology, 14(1):1-16. [26] Zuo J, Niu Q W, Frugis G, et al. 2002. The WUSCHEL gene promotes vegetative-to-embryonic transition in Arabi-dopsis[J]. The Plant Journal, 30(3): 349-359.
