杉木磷转运蛋白基因ClPht1;1的克隆及表达分析

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

【目的】PHT1基因家族是影响植物吸收磷营养的重要磷转运子之一。从杉木32号磷高效家系c DNA中克隆得到PHT1基因家族的1个杉木磷转运蛋白基因ClPht1;1,并对不同程度磷胁迫下ClPht1;1的时空表达进行研究,为杉木PHT1基因序列特征和功能结构的研究以及磷高效利用杉木基因型的选育奠定基础。【方法】根据PHT1基因家族序列保守性设计简并引物,以32号磷高效杉木基因型根系c DNA为模板进行扩增获得目的基因ClPht1;1的c DNA序列,使用RACE技术对目的基因进行全长克隆,并对其序列特征、同源性和编码磷转运蛋白结构进行分析。实时荧光定量PCR检测ClPht1;1在32号磷高效杉木家系根、茎、叶中的表达,检测中度缺磷胁迫下ClPht1;1在不同磷利用效率杉木4号、15号、25号、27号、28号、32号家系根系中的表达差异,以及在中度、重度缺磷胁迫下ClPht1;1在32号磷高效杉木家系根系中随时间序列的表达量变化。【结果】克隆得到1个杉木磷转运蛋白PHT1基因,命名为ClPht1;1(Gen Bank登录号:KX302006),基因序列编码区长1 638 bp,编码545 aa的蛋白质。ClPht1;1所编码蛋白质由12个疏水的跨膜区域组成,1个疑似跨膜域。每个跨膜结构域基本由17~25个氨基酸残基组成螺旋,同时跨膜蛋白的N端和C端均位于细胞质内,保守序列位于第4个跨膜域。构成蛋白质的主要骨架是α-螺旋,无信号肽序列。ClPht1;1基因编码蛋白与日本柳杉PHT基因编码蛋白的氨基酸序列相似性达到87.0%,与胡杨、油茶、马尾松等PHT家族基因编码蛋白的氨基酸序列相似性均在75%以上。ClPht1;1基因在杉木的根、茎、叶组织中均有表达,其中在根中的表达量最高,在叶中的表达量最低。在中度缺磷胁迫下,ClPht1;1基因在杉木不同家系根部的表达量为25号>27号>4号>15号>32号>28号。在中度和重度缺磷胁迫下,ClPht1;1基因在32号杉木家系根部的表达量随胁迫时间的延长而逐渐上升;恢复供磷后,ClPht1;1基因表达量逐渐下降至正常水平;重度缺磷胁迫下,ClPht1;1基因表达量要高于其在中度缺磷胁迫下的表达量。【结论】ClPht1;1基因具有PHT1基因家族的典型结构,其编码蛋白的氨基酸序列与日本柳杉等磷转运蛋白氨基酸序列具有高度相似性,为杉木高亲和磷转运蛋白PHT1基因家族成员。ClPht1;1基因主要在杉木的根部表达,在叶片中的表达量较低;杉木磷利用效率越强,ClPht1;1基因在其根部的表达量越高。在不同磷利用效率的杉木家系中ClPht1;1基因表达量存在较大差异。ClPht1;1基因的表达受低磷胁迫的诱导,缺磷胁迫下ClPht1;1基因表达量明显升高,恢复供磷后ClPht1;1基因表达量明显降低。

[ 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 ClPhtl; 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 ClPhtl; 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 ClPhtl; 1 in roots, stems and leaves of No. 32 C. lanceolata with high P-efficiency, differential expressions of ClPhtl; 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 etticiency under moderate P deficiency stress, and the CIPhtl; 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 PHTI gene was cloned and named ClPhtl; 1 （GenBank accession number KJ493165）. The coding region of the gene sequence was 1 638 bp, code 545 aa protein. ClPhtl; 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 c^-helix, no signal peptide sequence. The similarity of amino acid sequences of encoding proteins of ClPhtl; 1 was 87% with Cryptomeria japonica PHT gene, and above 75% with Populus, Camellia, Pinus massoniana etc. PHT family gene. CIPhtl; 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 CIPhtl; 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 ClPhtl; 1 in the root of No. 32 increased with the extension of stress time ; The expression level of CIPhtl; 1 was gradually decreased to normal level after the recovery of phosphorus supply; ClPhtl;1 gene expression in severe P deficiency stress was higher than that under moderate P deficiency stress. [ Conclusion ] ClPhtl; 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 PHTI gene family of C. lanceolata. ClPhtl;l 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 ClPhtl; 1 gene expression in its roots. There were large differences in the expression level of CIPhtl; 1 gene among families with different phosphorus use efficiencies. CIPhtl; 1 gene expression was induced by low phosphorus stress; Under P deficiency stress, the expression level of CIPhtl; 1 was significantly increased, and the expression of ClPhtl; 1 gene was significantly decreased after the recovery of phosphorus supply.