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Scientia Silvae Sinicae ›› 2017, Vol. 53 ›› Issue (5): 33-42.doi: 10.11707/j.1001-7488.20170505

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Cloning and Expression Analysis of Phosphate Transporter Gene ClPht1;1 in Cunninghamia lanceolata

Su Shuoshuo1, Li Ming1,2, Wu Pengfei1,2, Zhang Ying1, Ma Xiangqing1,2   

  1. 1. College of Forestry, Fujian Agriculture and Forestry University Fuzhou 350002;
    2. State Forestry Administration Engineering Research Center of Chinese Fir Fuzhou 350002
  • Received:2016-06-15 Revised:2016-08-12 Online:2017-05-25 Published:2017-06-22

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Abstract:

[Objective] PHT1 gene family is one of the important phosphorus transporters which affect the uptake of phosphorus in plants. A Cunninghamia lanceolata phosphate transporter gene of PHT1 gene family was cloned from No.32 phosphorus efficient C. lanceolata cDNA,and the temporal and spatial expression of ClPht1;1 under different levels of phosphorus stress was studied to lay the foundation for the research of PHT1 gene sequence characteristics, functional structure and the selection of the C. lanceolata genotype which is efficient in phosphorus utilization.[Method] According to the designed degenerate primer based on the conserved sequence of PHT1 gene family, the target gene ClPht1;1 cDNA sequence was obtained by amplification of the root cDNA of the P-efficient family No.32 of C. lanceolata as the template, the full length of the target gene was cloned by RACE technique, and analyzed its sequence characteristics, homology and protein structure were then analyzed. The real-time fluorescence quantitative PCR was used to detect the expression of ClPht1;1 in roots, stems and leaves of No.32 C. lanceolata with high P-efficiency, differential expressions of ClPht1;1 in the roots of C.lanceolata families of No.4,No.15,No.25,No.27,No.28 and No.32 in different P-utilization efficiency under moderate P deficiency stress, and the ClPht1;1 expression change with time in the root system of phosphorus efficient C.lanceolata No.32 under moderate and severe phosphorus deficiency stress.[Result]A C.lanceolata PHT1 gene was cloned and named ClPht1;1(GenBank accession number KJ493165). The coding region of the gene sequence was 1 638 bp, code 545 aa protein. ClPht1;1 encoded proteins were composed of 12 hydrophobic transmembrane domains, including 1 suspected transmembrane domains. Each transmembrane domain was composed of 17-25 amino acid residues, and the N and C ends of transmembrane proteins were located in the cytoplasm, and the conserved sequences were located in the fourth transmembrane domains. The main skeleton of the protein was α-helix, no signal peptide sequence. The similarity of amino acid sequences of encoding proteins of ClPht1;1 was 87% with Cryptomeria japonica PHT gene, and above 75% with Populus, Camellia, Pinus massoniana etc. PHT family gene. ClPht1;1 genes were expressed in root, stem and leaf of C.lanceolata, and the highest expression was in root,the lowest was in leaf; Under moderate P deficiency stress, the expression level of ClPht1;1 in the root of C.lanceolata varied among families in a order of No.25 > No.27 > No.4 > No.15 > No.32 > No.28. Under moderate and severe P deficiency stresses, the expression of ClPht1;1 in the root of No.32 increased with the extension of stress time; The expression level of ClPht1;1 was gradually decreased to normal level after the recovery of phosphorus supply; ClPht1;1 gene expression in severe P deficiency stress was higher than that under moderate P deficiency stress.[Conclusion] ClPht1;1 gene has the typical structure of PHT1 gene family, and its protein amino acid sequence is highly similar to the amino acid sequences of Cryptomeria japonica phosphate transporter protein, it is a member of the high affinity phosphate transporter PHT1 gene family of C.lanceolata. ClPht1;1 gene was mainly expressed in root of C.lanceolata, and its expression was lower in leaf. The stronger the phosphorus use efficiency, the higher the ClPht1;1 gene expression in its roots.There were large differences in the expression level of ClPht1;1 gene among families with different phosphorus use efficiencies. ClPht1;1 gene expression was induced by low phosphorus stress; Under P deficiency stress, the expression level of ClPht1;1 was significantly increased, and the expression of ClPht1;1 gene was significantly decreased after the recovery of phosphorus supply.

Key words: Cunninghamia lanceolata, phosphate transporter, PHT1;1, RACE, RT-qPCR, phosphorus use efficiency

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