The 5'UTR-intron of the Gladiolus polyubiquitin promoter GUBQ1 enhances translation efficiency in Gladiolus and Arabidopsis

Abstract

Background: There are many non-cereal monocots of agronomic, horticultural, and biofuel importance. Successful transformation of these species requires an understanding of factors controlling expression of their genes. Introns have been known to affect both the level and tissue-specific expression of genes in dicots and cereal monocots, but there have been no studies on an intron isolated from a non-cereal monocot. This study characterizes the levels of GUS expression and levels of uidA mRNA that code for β-glucuronidase (GUS) expression in leaves of Gladiolus and Arabidopsis using GUBQ1, a polyubiquitin promoter with a 1.234 kb intron, isolated from the non-cereal monocot Gladiolus, and an intronless version of this promoter.

Results: Gladiolus and Arabidopsis were verified by Southern hybridization to be transformed with the uidA gene that was under control of either the GUBQ1 promoter (1.9 kb), a 5' GUBQ1 promoter missing its 1.234 kb intron (0.68 kb), or the CaMV 35 S promoter. Histochemical staining showed that GUS was expressed throughout leaves and roots of Gladiolus and Arabidopsis with the 1.9 kb GUBQ1 promoter. GUS expression was significantly decreased in Gladiolus and abolished in Arabidopsis when the 5'UTR-intron was absent. In Arabidopsis and Gladiolus, the presence of uidA mRNA was independent of the presence of the 5'UTR-intron. The 5'-UTR intron enhanced translation efficiency for both Gladiolus and Arabidopsis.

Conclusions: The GUBQ1 promoter directs high levels of GUS expression in young leaves of both Gladiolus and Arabidopsis. The 5'UTR-intron from GUBQ1 resulted in a similar pattern of β-glucuronidase translation efficiency for both species even though the intron resulted in different patterns of uidA mRNA accumulation for each species.

Figure 1

A. Diagram of the DNA constructs used for transformation.Gladiolus GUBQ1promoter (G1-1) and an intronless version (G1-3). TS, transcription start; E, 5′UTR-exon; 5′U-I, 5′UTR intron; 35 S, CaMV 35 S promoter. Negative numbers represent the nucleotides on the 5′ side of the translation start codon. For Gladiolus transformation pUC-GUS was used as the backbone vector, pCAMBIA1391Z for Arabidopsis transformation, and pBI121-GFP for transient transformation of tobacco. B. Relative fold increase in the specific GUS activity for leaves from Arabidopsis (white bars) and Gladiolus plants (shaded bars) transformed with either the CaMV 35 S, G1-1, or G1-3 constructs. Three independently transformed lines were used for each of the constructs analyzed for GUS expression. Standard error shown.

Figure 2

DNA blots of genomic DNA isolated from (A)Gladiolusand (B)Arabidopsisplants containing theuidAgene under control of either the CaMV 35 S (35 S), G1-1, or G1-3 promoter. Genomic is digested with Hind III and hybridized with a 511 bp uidA specific probe. Numbers indicate the transgenic plant lines or non-transformed (NT) plant. Two size markers, 3 and 10 kb, are shown.

Figure 3

Histochemical GUS staining of transgenic (A)Gladiolusand (B)Arabidopsisplants containing theuidAgene under control of either the CaMV 35 S (35 S), G1-1 or G1-3 promoter. The Gladiolus plant material was taken three weeks after subculture from plantlets growing in vitro on MS medium. Plant tissue was incubated 16 h at 37°C in the staining solution. Magnification bars in A represent 2 mm.

Figure 4

Expression of theuidAgene in transgenicGladiolusorgans and callus. GUS specific activity was measured using three plants for each of the three transformed lines, and the mean and standard error are shown.

Figure 5

Expression of theuidAgene in transgenicGladiolusleaves. A. Relative uidA mRNA levels. B. Specific GUS enzyme activity. Each bar represents a transformed plant line in A and B. Lines from left to right-G1-1: 1, 2, 3 and G1-3: 4, 5, 6. The black bar represents the average of the plant lines for each construct. GUS specific activity and uidA mRNA levels were measured using three plants for each line. The mean and standard error are shown. C. Specific GUS enzyme activity/unit uidA mRNA. Average GUS activity was divided by its corresponding mRNA level for each DNA construct.

Figure 6

Expression of theuidAgene in transgenicArabidopsisleaves. A. Relative uidA mRNA levels. B. Specific GUS enzyme activity. Each bar represents a transformed plant line in A and B. Lines from left to right-G1-1: 113, 117, 118, G1-3: 131, 132, 134, 35 S: 1, 2, 7. The black bars represent the average of the plant lines for each construct. GUS specific activity and uidA mRNA levels were measured using three plants for each line, and the mean and standard error are shown. C. Specific GUS enzyme activity/unit uidA mRNA. Average GUS activity of each construct was divided by its corresponding mRNA level for each DNA construct.

Figure 7

Transient expression of thegfpgene in tobacco leaves. A. Relative GFP mRNA level. B. Relative amount of GFP expressed. C. Specific GFP amount/unit mRNA of GFP. Average GFP level of three leaves for each construct divided by its corresponding mRNA level. G1-1(A) contained the Arabidopsis actin7 gene and its 5′-UTR intron (540 bp) instead of the Gladiolus 5′-UTR intron.

Figure 8

RT-PCR analysis using the primers 5′UTR-exon F anduidA200 R/gfpR in (A) transgenicGladiolusleaves (B) transgenicArabidopsisleaves, and (C) transiently transformed tobacco leaves. Western blot analysis of GFP shown in bottom panel of C. The vectors were pUC-GUS for Gladiolus, pCAMBIA for Arabidopsis, and pBI121 for tobacco. pCAMBIA contains “LacZ alpha, truncated” between the GUBQ1promoter and uidA gene”. Abbreviations: Non-transformed plants (N), G1-1 (1), G1-3 (3), G1-1 promoter with act7 intron [1(A)], transient expression with pBI121 vector (−). Actin gene primers specific to each plant species analyzed were used as the reference gene to verify RT-PCR performance and the possibility of DNA contamination (bottom panel in A and B, middle panel in C).