GmPRP2 promoter drives root-preferential expression in transgenic Arabidopsis and soybean hairy roots

Abstract

Background: Promoters play important roles in gene expression and function. There are three basic types of promoters: constitutive, specific, and inducible. Constitutive promoters are widely used in genetic engineering, but these promoters have limitations. Inducible promoters are activated by specific inducers. Tissue-specific promoters are a type of specific promoters that drive gene expression in specific tissues or organs. Here, we cloned and characterized the GmPRP2 promoter from soybean. The expression pattern indicated that this promoter is root-preferential in transgenic Arabidopsis and the hairy roots of soybean. It can be used to improve the root resistance or tolerance to pathogens, pests, malnutrition and other abiotic stresses which cause extensive annual losses in soybean production.

Results: The GmPRP2 promoter (GmPRP2p-1062) was isolated from soybean cv. Williams 82. Sequence analysis revealed that this promoter contains many cis-acting elements, including root-specific motifs. The GmPRP2p-1062 and its 5'-deletion fragments were fused with the GUS reporter gene and introduced into Arabidopsis and the hairy roots of soybean to further determine promoter activity. Histochemical analysis in transgenic Arabidopsis showed that GUS activity was mainly detected in roots and hypocotyls in all deletion fragments except GmPRP2p-471 (a 5'-deletion fragment of GmPRP2p-1062 with 471 bp length). GUS activity was higher in transgenic Arabidopsis and hairy roots with GmPRP2p-1062 and GmPRP2p-852 (a 5'-deletion fragment of GmPRP2p-1062 with 852 bp length) constructs than the other two constructs. GUS activity was enhanced by NaCl, PEG, IAA and JM treatments and decreased by SA, ABA and GA treatments in transgenic Arabidopsis.

Conclusions: GmPRP2p-1062 is a root-preferential promoter, and its core fragment for root-preferential expression might lie between -369 and +1. GmPRP2p-852 may be useful in the genetic engineering of novel soybean cultivars in the future.

Figure 1

The expression level of GmPRP2 by qRT-PCR in different tissues. The relative expression level of GmPRP2 from real-time PCR was in different tissues. R, root; S, stem; L, leaf; F, flower; Sd, seed; H, hypocotyl. Data are the means of three replicates with SE shown by vertical bars. The capitals differ significantly by one-sided paired t test at P < 0.01.

Figure 2

Sequence of the GmPRP2p-1062 denoting the cis -elements predicted by the PLACE and PlantCARE databases and schematic diagrams of truncated GmPRP2p-1062 constructs. A A 1,236 bp 5’-flanking region fragment of the PRP2 gene, containing 1062 bp promoter sequence. The translated start site is defined as +1. The TATA box, partial CAAT box, Wbox, ABRE, MYB, MYC, root-specific elements are underlined with different colors. B Schematic diagrams of truncated GmPRP2p-1062 constructs. The numbers on the left indicate 5’-deletion fragments of the promoter. Some cis-elements are also marked with the colored columns.

Figure 3

GUS histochemical assays in T ₃ transgenic Arabidopsis seedlings carrying GmPRP2p-1062, GmPRP2p-852, GmPRP2p-471 and GmPRP2p-369 constructs. Photographs were taken 1 d, 3 d, 5 d, 7 d, 10 d, and 20 d from seeds placed from 4°C to 22°C on 1/2 MS plates.

Figure 4

GUS activity assays in T ₃ transgenic Arabidopsis roots and leaves carrying GmPRP2p-1062, GmPRP2p-852, GmPRP2p-471 and GmPRP2p-369 constructs. GUS activity assays in T₃ transgenic Arabidopsis roots and leaves carrying different deletion fragments of the GmPRP2p-1062. Each promoter fragment had three transgenic lines. P1062-9, P1062-14, and P1062-16 are the three transgenic lines carrying the GmPRP2p-1062 construct; P852-5, P852-9, and P852-30 are the three transgenic lines carrying the GmPRP2p-852 construct; P471-4, P471-6, and P471-7 are the three transgenic lines carrying the GmPRP2p-471 construct; P369-2, P369-3, and P369-6 are the three transgenic lines carrying the GmPRP2p-369 construct. Data are the means of three replicates with SE shown by vertical bars. **differ significantly by one-sided paired t test at P < 0.01.

Figure 5

GUS histochemical assays in T ₃ transgenic Arabidopsis seedlings carrying GmPRP2p-1062, GmPRP2p-852, GmPRP2p-471 and GmPRP2p-369 constructs in the reproductive stage. A, E, I, M Leaves of 30-day-old seedlings of transgenic plant grown on soil with GmPRP2p-1062, GmPRP2p-852, GmPRP2p-471, and GmPRP2p-369 constructs, respectively. B, F, J, N Leaves of 40-day-old seedlings of transgenic plant grown on soil with GmPRP2p-1062, GmPRP2p-852, GmPRP2p-471, and GmPRP2p-369 constructs, respectively. C, G, K, O Floral organs of transgenic plants with GmPRP2p-1062, GmPRP2p-852, GmPRP2p-471, and GmPRP2p-369 constructs, respectively. D, H, L, P Silique of transgenic plants with GmPRP2p-1062, GmPRP2p-852, GmPRP2p-471, and GmPRP2p-369 constructs, respectively.

Figure 6

GUS activity of transgenic Arabidopsis with different constructs (GmPRP2p-1062, GmPRP2p-852, GmPRP2p-471, GmPRP2p-369) in roots in responses to MeJA, IAA, SA, GA, ABA, NaCl, PEG. The GUS activity for each treatment was measured in 20-day-old seedlings from each of three independent transgenic lines. Transgenic seedlings were treated for 24 h. Control seedlings were treated with water. P1062-9, P1062-14, and P1062-16 are the three transgenic lines carrying the GmPRP2p-1062 construct; P852-5, P852-9, and P852-30 are the three transgenic lines carrying the GmPRP2p-852 construct; P471-4, P471-6, and P471-7 are the three transgenic lines carrying the GmPRP2p-471 construct; P369-2, P369-3, and P369-6 are the three transgenic lines carrying the GmPRP2p-369 construct. Data are presented as the means of three replicates with SE shown by vertical bars. *and** differ significantly by one-sided paired t test at P < 0.05 and P < 0.01, respectively.

Figure 7

GUS histochemical assays in transgenic soybean hairy roots. GUS staining of the transgenic cotyledon with hairy roots carrying 35S promoter construct, PC3P1 vector, GmPRP2p-1062 construct, GmPRP2p-852 construct, GmPRP2p-471 construct, and GmPRP2p-369 construct.

Figure 8

GUS activity of transgenic soybean hairy roots with different constructs (GmPRP2p-1062, GmPRP2p-852, GmPRP2p-471, GmPRP2p-369, 35S). Box plot analysis of GUS activities obtained in transgenic soybean hairy roots with different constructs (GmPRP2-1062, GmPRP2-852, GmPRP2-471, GmPRP2-369, 35S). GUS activity is given in nmol 4-MU min⁻¹ mg⁻¹ total protein. The lower boundary of each box denotes the 25^th percentile for each promoter, the solid line within each box denotes the 50^th percentile, the upper boundary of each box denotes the 75^th percentile. Two ends of the vertical line denote the maximum and minimum. Outlying data are indicated by empty black circles.