关键词: 棉花, 启动子, 功能分析, GhSLD1, 鞘脂delta8-去饱和酶

Abstract: 【Objective】Cotton fiber is the main economic product of cotton. It is the epidermal cells of the ovule outer integument through polar elongation and secondary wall thickening. As one of the longest plant cells, the cotton fiber cells are regarded as an ideal material in the study of plant cell growth and development. Identification of promoters specifically or preferentially expressed in fiber cells is of great significance for basic research on fiber development and molecular breeding for improving fiber traits. 【Method】In this study, we cloned the promoter of GhSLD1 gene, which is predominantly expressed in fiber cells. Through the PlantCARE website for promoter sequence analysis, we identified the important cis-regulatory elements contained in the cloned sequence. According to the distribution of some important cis-regulatory elements, the cloned promoter fragments were deleted at 5 '- end. A total of 4 promoter fragments were obtained and the corresponding plant expression vector was constructed. The constructed plant expression vectors were used for genetic transformation of tobacco and cotton. The transgenic plants were identified through molecular identification of transgenic tobacco and cotton. GUS activity in different tissues, organs and fiber cells of transgenic plants at different development stages was also investigated. 【Result】The longest promoter cloned was 2900 bp in length. In addition to there were a lot of transcription regulatory elements in the promoter, the sequence also contained multiple abscisic acid response elements, the elements essential for the anaerobic induction, methyl jasmonate response elements, brassinolide response elements, the elements involved in seed-specific regulation, the elements involved in defense and stress responsiveness, and MYB transcription factor binding sites. Four promoter fragments with a length of 2900 bp (GhSLD-P1), 2178 bp (GhSLD1-P2), 1657 bp (GhSLD1-P3) and 1232 bp (GhSLD-P4) were obtained by the 5'-terminal deletion, respectively. The transgenic tobacco plants were generated after confirmed by molecular identification. GhSLD-P1, GhSLD1-P2 and GhSLD1-P3 did not express in transgenic tobacco, while GhSLD-P4 is widely expressed, and the expression level of GhSLD-P4 was similar to that of CaMV 35S promoter. The different sequence between GhSLD1-P3 and GhSLD-P4 contained four abscisic acid response elements, two brassinolide response elements, and three MYB binding sites. These cis-regulatory elements may be associated with the non-expression of GhSLD1-P1, GhSLD1-P2, and GhSLD1-P3 promoters in transgenic tobacco. The transgenic cotton plants of GhSLD1-P2 were obtained after confirmed by molecular identification. GhSLD1-P2 predominantly expressed in transgenic cotton fibers, and its expression level was higher at the elongation stage (10-15 DPA) of fiber cells while lower in the early developmental stage (5 DPA) of fiber cells and the stage of secondary cell wall deposition (20-30 DPA). 【Conclusion】The GhSLD1-P4 promoter was a widely expressed promoter, and the GhSLD1-P2 promoter was a fiber predominant expression promoter, which was highly expressed during the elongation of fibers. It could be applied to the study on the gene function involved in cotton fiber development and molecular breeding for improving fiber traits.

Key words: cotton, promoter, functional characterization; GhSLD1, sphingolipid delta8- desaturase