Journal: Plant Direct

Article Title: The maize premature senesence2 encodes for PHYTOCHROME‐DEPENDENT LATE‐FLOWERING and its expression modulation improves agronomic traits under abiotic stresses

doi: 10.1002/pld3.295

Figure Lengend Snippet: Cloning and characterization of the Pre2 candidate gene. (a) Gene structure of the pre2 candidate gene, Zm00001d0053300. Exons and introns are represented by filled rectangles and thin lines, respectively. Insertions of a Mutator ( Mu ) in intron1 and a retrotransposon (RTE) in intron8 in the pre2‐1 and pre2‐2 mutant alleles are represented by triangles, respectively. The gene‐specific primers (GSPs) used in the PCR‐fingerprinting and RT‐PCR expression analyses are shown above the exons and arrows are pointing in their directions. (b) Full‐length cDNA of Pre2 amplified by RT‐PCR using 3′‐RACE. Lanes 1 and 2 are both represent the FL‐cDNAs amplified by using PHN137957 and PHN137958 GSPs from the 5′‐UTR of the pre2 candidate gene. (c) RT‐PCR of the pre2‐1 allele and its wild type sib using PHN137983 and PHN137985 GSPs from the exon1 and exon2 of the pre1 candidate gene, respectively. Three additional mature transcripts of ~301 bp, ~339 bp, and ~552 bp sizes with variable intensities were detected in the pre2‐1 allele as compared to only one functional transcript of ~179 bp in its WT sib. The blue and green regions of bars on the right side of Figure represent the predicted polypeptides of the differentially spliced mature transcripts resulted in frame shift (FS) and/or early termination (T) as compared to the functional predicted polypeptide represented by the black bars. Total RNA from ten days old seedlings was used in the RT‐PCR analysis. (d) Southern blot (SB) analysis of a segregating population detecting an RFLP of ~8.0 Kb/ EcoRI associated with the pre2‐2 mutant allele phenotype. The FL‐cDNA was used as a DNA probe for hybridization in SB analysis. (E) PCR‐fingerprinting of 7 F1 plants (lanes 2–8) of pre2‐1 and pre2‐2 allelic cross using PHN137983 in combination with Mu ‐TIR (upper lane in Figure ), GSP‐Exon8‐F2 with RTE‐R1 primer (middle lane in Figure ), and PHN137983 with PHN137985 (lower lane in Figure ). (f) The RT‐PCR expression analysis of the pre2‐2 mutant allele and its WT sib using GSP‐Exon8‐F2 and GSP‐Exon9‐R1 primers of the pre2 candidate gene. Two additional mature transcripts were detected in the pre2‐2 mutant allele as compared to a functional transcript of ~700 bp size in its WT sib. The colored bars on the right side of Figure are representing the predicted polypeptides as described in Figure . Amplification of the ZmActin1 was used as a control in RT‐PCR analysis

Article Snippet: Since only a few partial ESTs, representing 3′ end of the Zm00001d053300 gene, were found in both the public and Corteva Agriscience maize databases, we amplified and cloned ~4.0 kb full‐length cDNA (FL‐cDNA) by RT‐PCR using two GSPs from the 5′UTR of the pre2 candidate gene in 3′‐RACE (Figure ).

Techniques: Cloning, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Functional Assay, Southern Blot, Hybridization, Control