Abstract
Arginase is the only enzyme capable of producing urea in plants. This enzyme also contributes to many important biological functions during plant growth and development, such as seed development, root development and plant nitrogen using. The unique rice arginase gene OsARG is known to affect nitrogen use efficiency and is also associated with higher yields in rice. In this study, we transformed OsARG into upland cotton R18 by Agrobacterium-mediated genetic transformation and analyzed the function of OsARG in transgenic cotton. Two independent OsARG expression transgenic cotton lines, ARG-26 and ARG-38, were obtained via transformation. Southern blot analysis indicated that two copies and one copy of the OsARG gene were integrated into the ARG-26 and ARG-38 genomes, respectively. Enzyme activity and RNA transcription analysis revealed that the OsARG gene is highly expressed in cotton. The nitric oxide content and the morphology of ARG-26 and ARG-38 seedlings were both affected by expression of the OsARG gene. Field experiments indicated that the polyamine and nitrogen content increased by more than two-fold in the T3 generation plants of the transgenic cotton lines ARG-26-2, ARG-26-7, ARG-38-8, and ARG-38-11, as compared with the control plants. After harvesting cotton fibers grown in field conditions, we analyzed the quality of fiber and found that the fiber length was increased in the transgenic lines. The average cotton fiber length for all of the transgenic cotton lines was two millimeters longer than the fibers of the control plants; the average cotton fiber lengths were 31.94 mm, 32.00 mm, 32.68 mm and 32.84 mm in the ARG-26ARG-26-2, ARG-26-7, ARG-38-8 and ARG-38-11 lines, respectively, but the average fiber length of the control plants was 29.36mm. Our results indicate that the OsARG gene could potentially be used to improve cotton fiber length traits.
Highlights
The application of nitrogen (N) is an important practice in cotton production
Southern blot analysis indicated that two copies and one copy of the OsARG gene were integrated into the ARG-26 and ARG-38 genomes, respectively
A new ORF was constructed that replaced the transit peptide nucleotide sequence (60bp, 20AA) of the OsARG cloned the rice arginase gene OsARG (CDS) (HM369061, 1023bp, 340AA) with the mitochondrion transit peptide (Mtp) nucleotide sequence (63 bp, 21AA) of the Arabidopsis thaliana aldehyde dehydrogenase gene (AtALDH, ALDH2B7).The new OsARG ORF was inserted into the PUC19 plasmid using the PstI and XhoI sites
Summary
The application of nitrogen (N) is an important practice in cotton production. The quantity of nitrogen fertilizer applied and the nitrogen use efficiency of cotton plants both strongly influence cotton yield and fiber quality [1, 2], and these factors are known to influence plant defense responses to biotic and abiotic stress [3]. Plants mainly acquire nitrogen via three steps: uptake, assimilation, and remobilization. Nitrogen remobilization is an important factor influencing plant nitrogen use efficiency; high nitrogen remobilization efficiency can cause extra organic state nitrogen to be restored and reused in plant metabolism, a situation that prevents the conversion of this nitrogen into nitric oxide (NO) and other inorganic nitrogen forms that would be wasted. Nitrogen remobilization is mainly executed via arginine (Arg) metabolism. Arg metabolism has an important significance for processes including seed development, germination, and seedling growth and development. Arg content can account for 90% of the soluble nitrogen in the tender tissues of seedlings [6, 7, 8]
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