| Among the plant potassium transporter families,HAK/KUP/KT gene family has the most members,which plays an important role in participating in plant growth and development and improving plant adaptability to the environment.At present,the whole genome identification of HAK/KUP/KT gene family has been carried out in many plants,but the systematic study of wheat HAK/KUP/KT gene family has not been reported.In this study,the family gene was identified and analyzed by wheat database and related analysis software,and using molecular biology method to research the biological function of wheat HAK/KUP/KT gene family.The main findings are as follows:(1)Identification and analysis of the whole genome of TaHAKs family gene56 wheat HAK/KUP/KT genes(hereafter called TaHAKs)were identified by using the Hidden Markov Model of K+ transporter to search the recently published wheat genome database.The phylogenetic analysis results showed that the TaHAKs could be divided into four clusters(cluster I,cluster II,cluster III and cluster IV),among which cluster I and cluster II had the largest number of genes(73%).By analyzing the distribution of TaHAKs in the genome,it was found that the TaHAKs gene was uniformly distributed in three subgenomes of A(17),B(17),D(22),but their distribution on the chromosome group was not balanced.Chromosome group 2,3,5,6and 7 contained 9-12 gene sequences,but chromosome group 1 and 4 only respectively contained 3(5.4%)and 2(3.6%)gene sequences.The analysis of gene structure and protein structure showed that the TaHAKs family gene contained 3-10 exons / 2-9introns,and the family protein contained 10-14 transmembrane regions.The analysis of Conserved Motif indicated that the 56 TaHAKs proteins contained a total of 25 conserved motifs,and most Conserved Motifs was marked as K+_trans superfamily in the CDD of NCBI.Using wheat RNA-seq database to analyze the tissue expression of the TaHAKs gene family,and it was found that the expression levels of cluster I and cluster IV genes were low in all tissues except TaHAK1 a,TaHAK1 b,TaHAK4-7AS and TaHAK4-7DS,while the expression levels of cluster II and cluster III genes were high in all tissues except TaHAK2,TaHAK13 and TaHAK12-1AS.(2)Analysis of gene expression patterns of TaHAKs under abiotic stressqRT-PCR method was used to study the expression pattern of 9 TaHAKs genes under different stress conditions.The results showed that the expression levels of 9TaHAKs genes were up-regulated under low potassium stress.However,the expression patterns of different genes are different.The expression patterns of these genes can be divided into two types.The first type is characterized by rapid and continuous upregulation,including TaHAK1,TaHAK7,TaHAK18 and TaHAK23.The second type of expression pattern represented the transient upregulation of genes,which included strong upregulation of genes for a short time after potassium deficiency stress,followed by a rapid downregulation to the control level,and the low level of gene expression was unchanged for the remaining time of stress,including TaHAK2,TaHAK9,TaHAK11,TaHAK16 and TaHAK17.Under salt stress,the expression levels of TaHAK1,TaHAK11,TaHAK17,TaHAK18 and TaHAK23 were down-regulated,but the expression levels of TaHAK7,TaHAK9 and TaHAK16 were up-regulated.Under 20%PEG6000 drought stress,the expression levels of 8 TaHAKs were up-regulated,and the expression levels of TaHAK2,TaHAK7,TaHAK16,TaHAK17 and TaHAK18 were significantly up-regulated.Comparatively,TaHAK1,TaHAK9 and TaHAK11 were moderately upregulated.(3)The subcellular localization of TaHAKs proteinThe fusion expression vectors TaHAK7-2DS-GFP,TaHAK11-2DL-GFP,TaHAK17b-5DL-GFP and TaHAK22-2AL-GFP were transformed into tobacco,respectively,and observed the subcellular location of fusion protein in tobacco epidermis cells.The results showed that TaHAK17B-5DL-GFP fusion protein was only expressed on cell membrane,while TaHAK7-2DS and TaHAK22-5DL fusion protein were expressed in cell membrane,nucleus and cytoplasm,and the fluorescence signal of TaHAK11-2DL-GFP fusion protein was so weak that the exact subcellular localization can’t be observed.(4)The function analysis of TaHAKs in yeast mutantThe K+ transport activity of TaHAK7-2DS,TaHAK11-2DL,TaHAK17b-5DL and TaHAK22-2AL was tested in yeast mutant strain CY162,which is defective in K+uptake.The results showed that TaHAK7-2DS,TaHAK11-2DL and TaHAK22-2AL could not make CY162 grow on the AP medium of c(K+)≤1m M,indicating that these genes did not have the K+ transport activity under c(K+)≤1m M in CY162.However,TaHAK17b-5DL could make CY162 grow on AP medium with c(K+)=1m M,indicating that the gene had the K+ transport activity under c(K+)=1m M in CY162.(5)The function analysis of TaHAK17b-5DL in ArabidopsisTaHAK17b-5DL was transformed into Arabidopsis to analyse the K+ uptaking function in plants.The results showed that the root length and fresh weight of the transgenic lines of athak5 mutant and wild type Arabidopsis were significantly higher than control untransgenic lines under the condition of c(K+)≤0.01 m M,but there were no significant difference in root length and fresh weight between the transgenic lines and the control non-transgenic lines under the condition of c(K+)≥1m M,which indicated that TaHAK17b-5DL could promote K+ to be uptaken by plants at the condition of c(K+)≤0.01 m M. |
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