Isolation of an efficient actin promoter for use in rice transformation.

Abstract

We have characterized the 5' region of the rice actin 1 gene (Actl) and show that it is an efficient promoter for regulating the constitutive expression of a foreign gene in transgenic rice. By constructing plasmids with 5' regions from the rice Actl gene fused to the coding sequence of a gene encoding bacterial P-glucuronidase, we demonstrate Pthat a region 1.3 kilobases upstream of the Actl translation initiation codon contains all of the 5'-regulatory elements necessary for high-leve1 P-glucuronidase (GUS) expression in transient assays of transformed rice protoplasts. The rice Actl primary transcript has a noncoding exon separated by a 5' intron from the first coding exon. Fusions that lack this Actl intron showed no detectable GUS activity in transient assays of transformed rice protoplasts. Deletion analysis of the Act 7 5' intron suggests that the intron-mediated stimulation of GUS expression is associated, in part, with an in vivo requirement for efficient intron splicing.

The promoter of the barley aleurone‐specific gene encoding a putative 7 kDa lipid transfer protein confers aleurone cell‐specific expression in transgenic rice

“…Using this construct, the number of spots in immature aleurone layers increased substantially (Figure 4d). Still, compared with aleurone layers bombarded with the pActl-F construct (McEIroy et al, 1990), which contains the promoter of the constitu- To quantify Ltp2 promoter activity in particle bombardment experiments, a second control plasmid (Maas et al, 1991) containing the LUC gene under the control of the 355 promoter was co-bombarded with the Ltp2-Gus construct. Following incubation on tissue culture medium, protein was extracted from the grains in a buffer that allowed measurement of both LUC and GUS activity (for details, see Experimental procedures).…”

“…For the microprojectile bombardment expedments, the following constructs were used: pActl-F (McEIroy et al, 1990) containing the dce Actinl promoter fused to the uidA reporter gene encoding the GUS enzyme and the 3' non-coding region of the nopaline synthase gene; pRT-ex/s-int/s-LUC (Maas et aL, 1991) containing the 35S CaMV promoter plus the maize Sh I first exon/intron fused to the firefly luciferase gene and the pelyadenylation signal of CaMV 35S; pRT101C1 (Wienand, personal communication) containing the 35S CaMV promoter, the maize C1 cDNA (PazAres et al, 1987) and the pelyadenylation signal of CaMV 35S; pMF6Lc (Blowers, personal communication) containing the 35S CaMV promoter-Adhl intron, the Lc cDNA (Ludwig et al, 1989) and the 3' non-coding region of the nopaline synthase gene. The Ltp2 promoter contained on the 0.84 kb Bglll fragment (sequence presented in Figure 1) was inserted into the BamHI site of pBluescdpt in front of the GUS reporter gene and the 3' non-coding region of the nopaline synthase gene to give the Ltp2-Gus construct.…”

Cis-regulatory elements used to control gene expression in plants

“…However, some exceptions to this rule have been observed, for example, in Arabidopsis thaliana and Oryza sativa plants. Due to the function of actin (essential protein forming the cytoskeleton), actin promoters such as OsAct1 are constitutive, (McElroy et al 1990) but yet, their activity may take a different form when natural promoters are cloned into expression constructs. For example, the Act2 promoter, which is a modified Arabidopsis actin promoter, is substantially constitutive, but does not promote protein expression in hypocotyl, seed coat and microsporangia of Arabidopsis.…”

Impaired Function of the Tonoplast-Localized Sucrose Transporter in Rice, OsSUT2, Limits the Transport of Vacuolar Reserve Sucrose and Affects Plant Growth

“…First-strand cDNAs were used in RT-PCR with gene-specific primers and control primers for the housekeeping genes Act1 (McElroy et al, 1990) and OsUBQ5 (Jain et al, 2006). The gene-specific primers used were as follows: for OsSUT2, 5#-CTATCAT-GATGGTGTGAGAATG-3# and 5#-CTACCCAGTGACACAATAACCT-3#; for Act1, 5#-GGAACTGGTATGGTCAAGGC-3# and 5#-AGTCTCATGGATAC-CCGCAG-3#; and for OsUBQ5, 5#-GACTACAACATCCAGAAGGAGTC-3# and 5#-TCATCTAATAACCAGTTCGATTTC-3#.…”