The Expression And Regulation Of A High Affinity K~+ Transporter Gene From Aeluropus Littoralis

Potassium(K⁺) is a major essential mineral nutrient in higher plants.High-affinity K⁺ transporters play an important role in K⁺ absorption of plants.Crop physiological studies show that potassium is relative to resisting abiotic stress,but the knowledge of K⁺ transporters in molecular response to abiotic stress is poorly understood.AlHAK1 was cloned from Aeluropus littoralis.AlHAK1 was less affected by Na⁺,and the expression of AlHAK1 was induced by salt.Transgenic tobacco plants with constitutive expression of AlHAK1 could resist salt which indicated that AlHAK1 was relative to salt tolerant.Whether AlHAK1 is relative to abiotic stress,and regulated by signal substance is unknown.So we used qPCR to determine the expression patterns of AlHAK1 across a range of organs and observed whether AlHAK1 is differentially upregulated or downregulated in seedlings under abiotic stresses and phytohormone.In addition,plant promoter can regulate the gene transcription with environmental change,so much of the analysis was also in comparison with features of the promoter of AlHAK1.To identify the expression and regulation mechanisms,AlHAK1 promoter fused to the GUS reporter gene have been introduced to tobacco and onion epidermis cells to see the activity of the promoter.First,we established the real-time PCR assay for quantitative analysis of AlHAK1. According to the sequences of AlHAK1 andβ-actin(as a reference control),two sets of primers were designed.Subsequently,PCR conditions,including annealing temperature and concentration of primers were optimized.Relative quantitative standard curves were established and the repeatability of the assay was also evaluated.The results show that PCR efficiency of AlHAK1 andβ-actin was 1.09 and 1.04,respectively.The correlation coefficient was 0.996 and 0.997,respectively.Intra-assay and inter-assay coefficiency variation for both genes were less than 5%.The real-time PCR for AlHAK1 established in this study is simple, precise and repeatable for relative quantifying gene,and will provide useful methodological basis for understanding functions of AlHAK1,clarifying relationship between mRNA expression levels.The real time PCR results showed that AlHAK1 was expressed in root,stem and leaf of plant grown under 3mM K⁺conditions in hydroponic culture,and weaker expression was detected in the root.AlHAK1 was abundantly expressed in young tissues but it was not expressed in aging tissues.When treated with K⁺-starvation,salt stress,drought and UV,the expression of AlHAK1 increased,which showed that AlHAK1 was in response to these abiotic stress,and increased Ca²⁺ concentration substantially increase the transcript levels of AlHAK1 under 400 mM NaCl in roots.Moreover,AlHAK1 genes were also found to be differentially upregulated in roots subjected to treatments with ABA,MeJA,GA3,SA.To elucidate its upregulated mechanism at the transcriptional level,we isolated a 1356 bp promoter fragment of AlHAK1 by Anchor-PCR.A total of 108 putative cis-elements were found in the upstream region of these genes,of which a Ca²⁺-responsive cis-element,drought induced element,wounding responsive element,meristem expression element,and hormoneresponsive cis-elements were identified,suggesting regulation of AlHAK1 by these abiotic and hormone factors.Fragments of AlHAK1 promoter fused to the GUS reporter gene have been introduced to tobacco via Agrobacterium-mediated transformation.The GUS expression directed by AlHAK1 promoter was not observed in tobacco leaf,stem and root,but in pollen,and transgenetic tobacco does not respond to to NaCl,drought and K⁺-starvation,which indicated that the AlHAK1 promoter was tissue specific expression in dicotyledonous plant.We suppose that the expression of AlHAK1 promoters between monocotyledon and dicotyledonous plant may be different.As a next step,we performed Agrobacterium-mediated transformation of onion epidermis with a construct containing GUS gene under the control of the AlHAK1 promoter.The transient expression of GUS in onion epidermis was observed,and the HP1 fragment had the strongest activity.The different expression of GUS in tobacco and onion epidermis indicated that the expression of AlHAK1 promoter in monocotyledon and dicotyledonous plant may be different.