Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (19): 3712-3722.doi: 10.3864/j.issn.0578-1752.2023.19.002

• SPECIAL FOCUS: GENE FUNCTION AND BREEDING IN COTTON • Previous Articles     Next Articles

Cloning and Functional Characterization of the Promoter of GhSLD1 Gene That Predominantly Expressed in Cotton Fiber

LIU Fang(), XU MengBei, WANG QiaoLing, MENG Qian, LI GuiMing, ZHANG HongJu, TIAN HuiDan, XU Fan, LUO Ming()   

  1. Biotechnology Research Center, Southwest University/Key Laboratory of Biotechnology and Crop Quality Improvement, Ministry of Agriculture and Rural Affairs, Chongqing 400716
  • Received:2022-11-07 Accepted:2022-12-13 Online:2023-10-01 Published:2023-10-08
  • Contact: LUO Ming

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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 2 900 bp in length. In addition to 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 2 900 bp (GhSLD-P1), 2 178 bp (GhSLD1-P2), 1 657 bp (GhSLD1-P3) and 1 232 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

Fig. 1

Expression pattern of GhSLD1 gene in various organs of cotton and various developmental stages of fiber and ovule"

Fig. 2

Amplification of GhSLD1-P1 promoter M: DNA marker; GhSLD1-P1: Promoter of GhSLD1 gene"

Table 1

The cis-acting regulatory elements contained in the GhSLD1-P1 promoter"

序号No. 功能Function 序列Sequence 正链Strand+ 反链Strand-
Ⅰ 脱落酸响应元件 Abscisic acid responsiveness CACGTG/ACGTG 4
Ⅱ 厌氧诱导元件 Anaerobic induction AAACCA 4
Ⅲ 茉莉酸甲酯响应元件 MeJA-responsiveness CGTCA 1
Ⅳ 油菜素内酯响应元件 Brassinolide responsiveness CANNTG 7 3
V MYB结合位点 MYB binding site AACCTAA/TAACCA/ CAACCA/ TAACTG 5 2
VI 种子特异调控元件 Seed-specific regulation CATGCATG 1
VII 胁迫响应元件 Defense and stress responsiveness ATTCTCTAAC 1

Fig. 3

Construction of GhSLD1 promoter plant expression vector LB: The left border of T-DNA region; RB: The right border of T-DNA region; NPTII: Kanamycin resistance gene; GUS: β-Glucuronidase gene (reporter gene); Ter: Terminator; CaMV35S: The 35S promoter of Cauliflower Mosaic Virus (CaMV)"

Fig. 4

GhSLD1 promoter transgenic tobacco amplification validation A: GhSLD1-P1 transgenic tobacco; B: GhSLD1-P2 transgenic tobacco; C: GhSLD1-P3 transgenic tobacco; D: GhSLD1-P4 transgenic tobacco. M: DNA Marker; +: Positive control, the promoter vector of the corresponding fragment is used as the amplification template; -: Negative control, the wild type tobacco gDNA is used as the amplification template, H2O: A blank control, and water is used as a PCR amplification template; and 1-4: The corresponding fragment of the promoter transgenic tobacco plant"

Fig. 5

Characteristics of expression of GhSLD1 promoters in basic tobacco tissues CaMV 35S: The transgenic tobacco plants of CaMV35S promoter; GhSLD1-P1-GhSLD1-P4: The transgenic tobacco plants of GhSLD1-P1, GhSLD1-P2, GhSLD1-P3, and GhSLD1-P4, respectively. A: Stigma, B: Stamen, C: Leaf, D: The cross-section of the petiole, E: The cross- section of the stem, F: Longitudinal section of the stem, G: Petal, H: The ovary on the day of flowering, I: Root"

Fig. 6

Identification of GhSLD1-P2 transgenic cotton M: DNA Marker; +: Positive control, the plant expression vector plasmid of GhSLD1-P2 was used as a template; C: Negative control, the genome DNA of non-transgenic cotton was used as a template; W: Empty control, H2O was used as template; 1-4: Transgenic cotton line 1-4"

Fig. 7

The activity of GUS in leaf and stem of transgenic cotton plants Non-T: Non-transgenic cotton, as a negative control; 8DP2-GUS: The transgenic cotton plants of pBI121-GhSLD1-P2::GUS vector; 35S-GUS: The transgenic cotton plants of pBI121-CaMV35S::GUS vector, as a positive control. The same as below"

Fig. 8

The activity of GUS in flower of transgenic cotton plants"

Fig. 9

The activity of GUS in ovules and fiber cells of transgenic cotton plants at various developmental stages 5-30 DPA: The ovules and fiber cells at 5-30 day post anthesis"

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