Parsley ubiquitin promoter displays higher activity than the CaMV 35S promoter and the chrysanthemum actin 2 promoter for productive, constitutive, and durable expression of a transgene in Chrysanthemum morifolium

Abstract

The chrysanthemum (Chrysanthemum morifolium) is one of the most popular ornamental plants in the world. Genetic transformation is a promising tool for improving traits, editing genomes, and studying plant physiology. Promoters are vital components for efficient transformation, determining the level, location, and timing of transgene expression. The cauliflower mosaic virus (CaMV) 35S promoter is most frequently used in dicotyledonous plants but is less efficient in chrysanthemums than in tobacco or torenia plants. Previously, we used the parsley ubiquitin (PcUbi) promoter in chrysanthemums for the first time and analyzed its activity in transgenic calli. To expand the variety of constitutive promoters in chrysanthemums, we cloned the upstream region of the actin 2 (CmACT2) gene and compared its promoter activity with the 35S and PcUbi promoters in several organs, as well as its durability for long-term cultivation. The CmACT2 promoter has higher activity than the 35S promoter in calli but is less durable. The PcUbi promoter has the highest activity not only in calli but also in leaves, ray florets, and disk florets, and retains its activity after long-term cultivation. In conclusion, we have provided useful information and an additional type of promoter available for transgene expression in chrysanthemums.

Keywords: Chrysanthemum morifolium; gene expression; parsley; promoter activity.

Fig. 1

Construction and activity of 35S pro::GUS, CmACT2 pro::GUS, and PcUbi pro::GUS transformants. (A) Expression level of actin genes in different organs. Expression of CmACT1 (upper) and CmACT2 (middle) were confirmed by RT-PCR. A house keeping gene CmTBP2 (lower) was used as a reference. (B) Schematic diagram of GUS-expression constructs to generate transformants. GUS: β-glucuronidase. HSPT: terminator of heat shock protein. (C) GUS activity was measured in calli at 3-months post-transformation. GUS activity was expressed as pmol 4MU mg⁻¹ protein min⁻¹ and shown transformed as a common logarithm. Circles indicate the value of GUS activity from independent lines; “mean” indicates mean GUS activity in each sample; “n” indicates the number of transgenic calli examined in this study.

Fig. 2

GUS activity in leaf tissue of 35S pro::GUS, CmACT2 pro::GUS, and PcUbi pro::GUS transformants. GUS staining in upper leaves (A) and GUS activity in mature leaves (B) at 10–11 months post-transformation was analyzed. For histochemical analysis, stain intensity and pattern varied among transformants, and representative samples are shown (A). GUS activity was measured in the mature leaves of each transformant line and compared with the staining results of upper leaves of the same line (B). GUS activity is expressed as pmol 4MU mg⁻¹ protein min⁻¹. Upper leaf staining results are indicated below the graph. Vertical bars in the graph show mean values. Highlighted samples expressed >100 pmol 4 MU mg⁻¹ protein min⁻¹ GUS activity and are considered GUS-positive.

Fig. 3

GUS activity in the flower tissue of 35S pro::GUS, CmACT2 pro::GUS, and PcUbi pro::GUS transformants. Histochemical staining of ray florets and disk florets (A). Stain intensity and pattern varied among transformants, and representative samples are shown. GUS activity of disk florets and ray florets at 10–11-months post-transformation is shown (B). GUS activity is expressed as pmol 4MU mg⁻¹ protein min⁻¹. Black bars: ray florets. Gray bars: disk florets. Vertical bars in the graph and following numbers are mean values of ray florets (black) and disk florets (gray). Highlighted samples expressed >100 pmol 4MU mg⁻¹ protein min⁻¹ GUS activity and are considered GUS-positive.

Fig. 4

GUS activity in long-term-cultivated 35S pro::GUS, CmACT2 pro::GUS, and PcUbi pro::GUS transformants. Positions of sample collection are schematically illustrated (A). Long-term-cultivated tips were obtained from plants that flowered once under short-day conditions, after which the main stem was cut and grown under long-day conditions for 2 years. The planted tips were prepared as for the long-term-cultivated tips, and other parts of the same plant were cut and transferred to fresh soil. These cuttings were harvested after 3–4 weeks of long-day conditions. GUS activity of the shoot tips in the 35S pro::GUS, CmACT2 pro::GUS and PcUbi pro::GUS transformants (B). Black bars: long-term-cultivated tips. Gray bars: planted tips. GUS activity is expressed as pmol 4MU mg⁻¹ protein min⁻¹. Vertical lines and following numbers show the mean of long-term-cultivated tips (black) and planted tips (gray). Highlighted samples expressed >100 pmol 4MU mg⁻¹ protein min⁻¹ GUS activity and are considered GUS-positive.