Cloning And Functional Analysis Of The Wheat Genes Related To The Root Low Phosphorus Stress Response At Seedling Stage

By understanding the mechanisms of wheat for adapting to the low phosphorus stress and finding the key genes envolved in the mechanisms, molecular breeding or molecular-assisted breeding have been important strategies for keeping the wheat high yield and good quality.We employed Zhengmai9023 as the starting material,took use of the Affymatrix gene chip technology to look for the differently expressed genes in wheat root under low phosphorus stress.We cloned four genes related to low phosphorus stress and conducted the biochemical function analysis with bioinformatics and qRT-PCR.1. Cloning of the selected differently expressed genes and their sequence analysisOn the basis of the selected datas of Affymatrix gene chip and Transcriptome sequence of Triticum urartu. we have cloned four genes through the way of RT-PCR and named them respectively:TaGST-like （F441468）. TaSPX3 （F194210）, TaPSI 1 （F290511）、TaPreoxidase-like （F451598）. The ORF of TaGST-like is 699-bp,which codes polypeptide consisting of 232 amino acids.The protein structure of TaGST-like has the N-terminal Thiordoxin domain and the C-terminal GST domain, This gene may play an important role in cleaning free radical produced by low phosphorus stress. The full length of TaSPX3 is 789-bp which codes a polypeptide of 262 amino acids, this gene is named TaSPX3 because its protein has high degree of sequence homology with OsSPX3isolated from rice. NCBI blasting showed the coding product of TaSPX3 has characteristin of SPX near the N-terminal,This gene has two introns and three exons, The components forcasting of TaSPX3 promoterpresentedthat this gene’s promoter region has two P1BS regions（-222-bp,-423-bp）, which could be combind by transcription factor PHR1.This gene may play an important role in the low phosphorus stress signal transduction passway.The ORF of TaPSI 1 is 243-bp, which encodes 80 animo acids. the function is unkown,its functioal role remained further research.The full length of TaPreoxidase-like is 360-bp, which codes 119 animo acids.This protein has a C-terminal conserved domain of Preoxidase superfamily.This gene may be induced at the early stage of the wheat root is stressed by low phosphorus.The detailed functions of these four candidate genes need more researches in the future.2. Expression analysis of low phosphorus stress related genesThe expression of the four candidate genes are detected by real-time RT-PCR in roots of eight different genotypes of wheat including Zhengmai₉023, Xinong979, Jimai17, Xiaoyan54, Xiaoyan6, Yunong202, Han6172, Zhongguochun.These wheats were cultured in low phosphate medium and normal phosphate medium.The results showed that TaGST-like was only up-regulated in low-P for YN202, and almostly down-regulate in low-P for all other genotypes of wheat, especially in XY54 and XY6.TaSPX3 wasup-regulated in low-P for all eight genotype s of wheat, this gene may play the same role in eight genotypes of wheat.TaPSI 1 and TaPreoxidase-like were up-regulated in low-P for JM17 and YN202, and expressed consistly in two Pi regimes for other genotypes of wheat. All these four gene were up-regulated in low-P for JM17 and YN202but differed in expression levels.This phenomenon may be related to the alternations in the subtle mechanisms of Pi regulatory networks in different genotypes of wheat.Semi-quantitative RT-PCR was used for tissue-specific expression analysis with actin gene as the inner control.TaSPX3 was induced positively by low phosphorus stress at in Zhengmai9023 root and shoot cultured for 14 days.TaPreoxidase-like was only highly expressed in root, and didn’t be induced by low-P. The two other candidate genes didn’t be detected in ZM9023 shoot and root.3. TaSPX3 protein prokaryotic expressionFor the further functional study of TaSPX3, we constructed recombinant vector pET-32a-TaSPX3. After sequencing verification, under IPTG induction,TaSPX3 fusional protein was obtained from prokaryotic expression cells. Fusional protein existed in the form of inc lusion body. For future study, we need to get the soluble fusional protein through the way of purification, denaturing and renaturing the inclusion body. Soluable fusion protein can be used for targeting its interaction proteins, determining its celluar localization.