A cotton-fiber-associated cyclin-dependent kinase a gene: characterization and chromosomal location

Abstract

A cotton fiber cDNA and its genomic sequences encoding an A-type cyclin-dependent kinase (GhCDKA) were cloned and characterized. The encoded GhCDKA protein contains the conserved cyclin-binding, ATP binding, and catalytic domains. Northern blot and RT-PCR analysis revealed that the GhCDKA transcript was high in 5-10 DPA fibers, moderate in 15 and 20 DPA fibers and roots, and low in flowers and leaves. GhCDKA protein levels in fibers increased from 5-15 DPA, peaked at 15 DPA, and decreased from 15 t0 20 DPA. The differential expression of GhCDKA suggested that the gene might play an important role in fiber development. The GhCDKA sequence data was used to develop single nucleotide polymorphism (SNP) markers specific for the CDKA gene in cotton. A primer specific to one of the SNPs was used to locate the CDKA gene to chromosome 16 by deletion analysis using a series of hypoaneuploid interspecific hybrids.

Figure 1

Diagrammatic comparison of cotton GhCDKA gene (a) with Arabidopsis thaliana AtCDKA; 1 Gene ((b), Genbank GI: 18408695). ATG represents the start codon. TGA and TAG are stop codons. The exons containing the coding regions are boxed (numbers 1–8). The exons located in the 5′-UTR region are represented by the L boxes. The positions of codons at the 5′ and 3′ ends within exons are indicated. Intron sizes are indicated under the intron lines.

Figure 2

Phylogenetic analysis of eleven plant CDKA proteins. The phylogenetic tree was constructed based on amino acidsequences of 11 plant CDKAs using the Clustal method. The eleven CDKA proteins are GhCDKA (Gossypium hirsutum); PtCDKA (Populus tremula x Populus tremuloides); HaCDKA (Helianthus annuus); PaCDKA (Picea abies); LeCDKA1 (Solanum lycopersicum); PncCDKA (Pinus contorta); CrCDKA (Chenopodium rubrum); HtCDKA (Helianthus tuberosus); AmCDKA (Antirrhinum majus); NtCDKA (Nicotiana tobacum); AtCDKA; 1 (Arabidopsis thaliana).

Figure 3

(a) Northern analysis of GhCDKA expression in different cotton tissues. Ten μg of total RNA from fibers (5, 10, 15, and 20 DPA, lanes 1–4), flowers (lane 5), leaves (lane 6), and roots (lane 7) was electrophoresed on an agarose gel, transferred onto a nylon membrane, and hybridized with a ³²P-labeled GhCDKA cDNA. Two EtBr-stained rRNA bands indicate that an equal amount of total RNA was loaded for each sample. The relative GhCDKA transcript levels were determined by the ratio of hybridized intensity of the 1.2 kb GhCDKA mRNA to the EtBr stained 28S rRNA band using the program of Scion Image for Windows (Scion Corporation). (b) RT-PCR analysis of GhCDKA mRNA. Total RNA from leaves, flowers, roots, and 5, 10, and 15 DPA fibers was used as template in generating first strand cDNA. Each cDNA was made 1x, 5x, 10x, and 20x dilutions and used as template for PCR amplification with two GhCDKA gene specific primers: CDKC-2 and CDK5-1.

Figure 4

Western blot analysis of GhCDKA. Seventy μg of total protein was subjected to SDS-PAGE, blotted onto a nitrocellulose membrane, and probed with anti-PSTAIRE antibody. Proteins samples are from 5 DPA (lane 1), 10 DPA (lane 2), 15 DPA (lane 3), and 20 DPA (lane 4) fibers, flowers (lane 5), and leaves (lane 6).

Figure 5

Alignment of 5′ flanking sequences of CDKA gene of DES119 (G. hirsutum), TM-1 (G. hirsutum), 3–79 (G. barbadense), and CMD11 (G. tomentosum) showing the presence of several SNPs. The arrow indicated the position and the direction of the SNP primer specific to the CDKA gene.

Figure 6

The electropherograms of two allelic SNPs, designated here as CDKA^cg (black) and CDKA^at (green), that corresponded to the polymorphism between G. hirsutum inbred TM-1 and G. barbadense-doubled haploid 3–79. Genomic dosage profiles are shown for [A] TM-1; [B] 3–79; [C–E] three hypoaneuploid-interspecific G. hirsutum x G. barbadense F₁ hybrids, [C] lacking G. hirsutum chromosome 4 (H4), [D] a monotelodisomic-16sh F1 (Te16SH) lacking most of the long arm of G. hirsutum chromosome 16, [E] a monotelodisomic 16 Lo F₁ (Te16LO) lacking most of the short arm of G. hirsutum chromosome-16; [F] a backcross disomic substitution plant (CSB-16) in which chromosome 16 of 3–79 has replaced the TM-1 chromosome 16; [G] a G. arboreum (A2 species); and [H] a G. raimondii plant (D5 species). Electropherograms revealing both peaks that indicate heterozygosity for both parental SNPs, as in the H4 interspecific F1 hybrid, indicate the locus is not in this chromosome. In contrast, absence of the G. hirsutum SNP CDKA^cg from CSB 16 and Te16SH hybrids and its presence in Te16LO concordantly indicate that the SNP marker is located in the long arm of the chromosome 16.