Molecular Cloning, Characterization And Evolution Of A Novel AP2/ERF Gene Differentially Expressed In Tetraploid Cotton

As a major source of fibers, cotton is an important economic crop and plays an important role in the global economy. With the development of textile technology and social demand, it is urgent to breed and plant cotton varieties with high yield production and super fiber quality. Hybrid cotton could greatly increase lint yield, modestly improve fiber quality and raise tolerance to stresses. Accordingly, hybrid cotton could create remarkable economic and social benefits. The research of the cotton hererosis is still the hot and important basic theory problem. The regulations in the transcription level are the main method of eukaryotic genes’ modulation, that effect and control many biological processes, and genes differently expressed between hybrids and parents may have relation to the transcript factors. So the studying in the genes which encoded the transcript factors and differently expressed between hybrids and parents will contribute to reveal the relationship between the regulating gene differently expressed and the hererosis, Many study have found that the transcript factors in ERF family have very important effects in the responses to biotic and abiotic stresses, they may helpful to make the plants to adapt to the changful environments and provide the selection predominance in plants evolution.In this research, an EST which is differently expressed between high yield upland cotton hybrids and parents in the seedling stage and has highly identification with ERF gene,was used as a template to isolation and characterization ERF gene in cotton. Through further analysis to discussion the relationship between this gene and the yield-components and explain the effect of this gene in the hererosis at the level in regulation of transcription.The mainly results are as follows:(1) Homologous cloning was performed to isolation the GhERF7 used a differently expressed EST as a template. The full length of this gene was 884bp, ORF was 58bp encoding 195 aa with 115bp 5’-UTR and 141bp 3’-UTR. The protein molecular weight was 22.1 kDa and a calculated pⅠwas 5.90. It had a typical AP2/ERF DNA domain (59 aa) between 70-128 in the aa position. Expression profile analysis found that this gene expressed in all of the vegetative organs, but highest expressed in the stems. Furthermore, it had higher expression in fibers initiation and enlongation periods (0-6 DPA), but nearly undetectable in fibers (10-15 DPA) and seeds (30 DPA).(2) Blast and homologous analysis found that GhERF7 had highly identification (91.4% in the full length of aa level) with GhERF1, and they were classified in the same branch (B3 subgroup) in ERF subfamily. Southern blot found both GhERFl and GhERF7 had two copies in tetraploid cotton genome. Genomic origin analysis revealed that GhERF1 and GhERF7 were homoeologous genes in the allotetraploid cotton (AD genome) with GhERF7 derived from the A subgenome and GhERFl from the D5 subgenome. GhERFl was undetected in the cotyledon and there was no distinctively different of its expression in the hybrids and two parents’leaves of the seedling, budding and full opening flower stage.Compared to the expression of GhERF1, GhERF7 was also ndetected in the cotyledon, but in other periods, GhERF7 was highly expressed in female parent, very lower in hybrids and no expression in male parent. The 5’ upstream region of GhERFl and GhERF7 was amplified by TAIL-PCR method,829bp and 978bp fragment of they were obtained respectively. Sequences analysis found they had many important cis-acting elements.(3) Sequences compared and homologous analysis found that there presented difference of the GhERF7 gene in diploid and allotetraploid cotton, but the homologous of the GhERF7 gene sequences between them reached 99%. According to the ampilification of GhERF7 in 81 upland cotton varieties and the pedigree analysis of these varieties, we found that GhERF7 gene was mainly exised in DPL and the derived varieties, but not exised in Stoneville and the derived varieties.(4) Using the F2 mapping population derived from the hybridization between upland cotton standard line TM-1 and the sea-island cotton cultivar Hai7124, GhERFl was localized on chromosome 16 (D7). The positions of GhERF7 in the immortalized F2 genetic map of XZM2 and the BC1 mapping population derived from [(TM-1 x Hai 7124) x TM-1] were the same, localized on chromosome 7 (A7) between the SSR marker NAU1043 and NAU3810. In two environments, each had a QTL for boll number also in this region of GhERF7 to NAU3810. Single marker analysis found that this gene had distinct relationship to yield-components’ other factors (seed-yield, lint-yield, lint percentage, lint index and so on).