Molecular cloning and functional analysis of ScHAK10 gene promoter from sugarcane (Saccharum officinarum L.)

Abstract

Transcriptional regulation of the high-affinity K+ (HAK) transporter gene is an important mechanism of plant response to low potassium stress. Cloning and analysis of the promoter of potassium transporter gene is not only helpful to its expression pattern and regulation mechanism but also important to improve the potassium absorption efficiency in sugarcane. The potassium transporter gene ScHAK10 is highly expressed and induced by low potassium stress in sugarcane, but the functions of its promoter is still unclear. In the present article, the 1918 bp promoter region of the ScHAK10 gene (pScHAK10) was cloned by genomic walking technique. Computational analysis affirmed the existence of abiotic stress-responsive cis and core cis-elements, such as TATA box, CAAT box, phytohormone responsive, stress response and light response motifs. GUS histochemical staining of transgenic Arabidopsis thaliana seedlings showed that the leaf, corolla, and root tip were deeply stained, and paraffin sections of root tip tissue showed GUS staining in ductal tissue of A. thaliana shallowly stained. The 5′-terminal deletion of the promoter was cloned, and the lengths of 1918 (full), 1623 (Q1), 1332 (Q2), 957 (Q3), 576 (Q4), and 357 bp (Q5) were cloned into the GUS reporter vector for A. thaliana transient transformation. The transgenic plants generated through a single event exhibited a promising expression of the GUS reporter protein, which was treated with salt, low potassium, IAA and cold stress conditions. The results showed that the promoter activity correlates with the promoter fragment's length, and the long promoter fragment exhibits higher training. The Q5 was the least active and could not drive GUS expression. Under abiotic stress, the expression of GUS enzyme activity varies among different promoter fragments. Under low potassium and high salt stress, Q3 and Q4 showed the highest promoter activity. The Q1 and Q4 led the highest promoter activity during IAA and cold stress. These findings help to understand the molecular mechanism of ScHAK10 expression regulation and provide a theoretical basis for improving potassium uptake efficiency and breeding potassium-efficient varieties in sugarcane.