杉木磷转运蛋白基因ClPht1;1的克隆及表达分析

发布时间：2018-10-08 12:32

【摘要】：【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。
[Abstract]:[objective] PHT1 gene family is one of the important phosphorus transporters affecting plant phosphorus uptake. A phosphorous transporter gene (ClPht1;1,) of Chinese fir (Cunninghamia lanceolata) was cloned from c DNA of Chinese fir 32, and the temporal and spatial expression of ClPht1;1 was studied under different phosphorus stress. The results laid a foundation for the study of PHT1 gene sequence and functional structure and the selection of Chinese fir genotypes with high phosphorus efficiency. [methods] degenerate primers were designed according to the conserved sequence of PHT1 gene family. The c DNA sequence of the target gene ClPht1;1 was amplified from the root system of Chinese fir genotypes 32, and the target gene was cloned by RACE technique, and its sequence characteristics were analyzed. Homology and structure of phosphorous transporter were analyzed. Real-time fluorescence quantitative PCR was used to detect the expression of ClPht1;1 in roots, stems and leaves of Chinese fir families with high phosphorus efficiency, and the difference of ClPht1;1 expression in the roots of Chinese fir families 4, 15, 25, 27, 28, 32 under moderate phosphorus deficiency stress. And under moderate and severe phosphorus deficiency stress, the expression of ClPht1;1 in the roots of Chinese fir family No. 32 was changed with time series. [results] A phosphorous transporter PHT1 gene was cloned from Chinese fir (Cunninghamia lanceolata). Named ClPht1;1 (Gen Bank accession number: KX302006), the coding region of the gene sequence was 1 638 bp, encoding 545 aa protein. The protein encoded by ClPht1 1 was composed of 12 hydrophobic transmembrane regions and a suspected transmembrane domain. Each transmembrane domain consisted of 17 ~ 25 amino acid residues, and the N-terminal and C-terminal of the transmembrane protein were located in the cytoplasm, and the conserved sequence was located in the fourth transmembrane domain. The main skeleton of the protein is 伪 -helix. The amino acid sequence similarity between the unsignaled peptide sequence. ClPht1 gene encoding protein and the PHT gene protein of Japanese willow tree is 87.0, which is similar to the amino acid sequence of Populus euphratica and Camellia oleifera. The amino acid sequence similarity of PHT family proteins of Pinus massoniana was more than 75%. ClPht1 gene was expressed in the roots, stems and leaves of Chinese fir, and the highest in roots and the lowest in leaves. Under moderate phosphorus deficiency stress, the expression of ClPht1 gene in the roots of different Chinese fir families was No. 27, No. 15, No. 32, No. 28. Under moderate and severe phosphorus deficiency stress, the expression of ClPht1 1 gene in the roots of Chinese fir family No. 32 gradually increased with the extension of stress time, and the expression of ClPht1 gene decreased gradually to normal level after phosphorus supply was restored. The expression of ClPht1;1 gene in severe phosphorus deficiency stress was higher than that in moderate phosphorus deficiency stress. [conclusion] ClPht1;1 gene has the typical structure of PHT1 gene family. The amino acid sequence of the encoding protein is highly similar to that of the phosphorous transporter such as the Japanese willow fir. It is a member of the PHT1 gene family of high affinity phosphorous transporter of Chinese fir, which is mainly expressed in the root of Chinese fir (Cunninghamia lanceolata). The higher the phosphorus use efficiency, the higher the expression of ClPht1 1 gene in the roots of Cunninghamia lanceolata (Cunninghamia lanceolata). In Chinese fir families with different P use efficiency, the expression of ClPht1;1 gene was significantly different. The expression of ClPht1 gene was induced by low phosphorus stress, and the expression of ClPht1;1 gene increased significantly under phosphorus deficiency stress, and the expression of ClPht1;1 gene decreased significantly after phosphorus recovery.
【作者单位】：福建农林大学林学院;国家林业局杉木工程技术研究中心;
【基金】：国家自然科学基金海峡联合基金项目(U1405211) 国家林业局杉木工程技术研究中心科技成果孵化基金(6213C0111)
【分类号】：S791.27

【参考文献】