杉木磷转运蛋白ClPht1;2基因克隆与表达特性分析

Cloning and Expression Analysis of Phosphorus Transporter Gene ClPht1;2 in Cunninghamia lanceolata

磷酸转运蛋白PHT1家族是介导植物磷素吸收与转运分配的重要基因家族。从第3代杉木优良无性系洋061中克隆获得1个杉木磷转运蛋白ClPht1;2,并对不同程度磷胁迫下ClPht1;2的时空表达进行分析,为了解杉木磷转运蛋白基因结构和功能表达奠定基础。以转录组测序获得的ClPht1;2核心序列为基础,以杉木洋061无性系根系cDNA克隆为模板,利用cDNA末端快速扩增技术(RACE)克隆目的基因的全长,采用实时荧光定量PCR技术,检测了杉木洋061无性系在不同组织中ClPht1;2的表达,以及磷饥饿诱导3、10、25 d下ClPht1;2在根中的表达量变化。克隆得到1个杉木PHT1家族基因ClPht1;2(GeneBank登录号:MK450598)。ClPht1;2编码氨基酸序列与日本柳杉磷转运蛋白家族基因编码氨基酸序列相似性为93%,与马尾松、油茶、毛果杨等植物磷转运蛋白家族基因的编码氨基酸序列比对,结果相似性均>72%。ClPht1;2基因序列编码区长1565 bp,编码511个氨基酸,蛋白理论分子量60.024 ku,为疏水蛋白,不具有信号肽,潜在磷酸化位点42个。ClPht1;2所编码蛋白质由12个跨膜结构组成,其中11个为确定跨膜域,多肽链中α螺旋占42.65%,无规则卷曲占42.11%,延伸链占15.25%。ClPht1;2在杉木洋061无性系的根、茎、叶中均有表达,在叶片中的表达量最高,在根中的表达量最低。与正常磷供应相比,根系ClPht1;2的表达水平在低磷胁迫10 d时显著增加,到25 d时下降至正常磷供应时的表达水平;ClPht1;2在无磷胁迫处理3 d时表达量降低,在10 d时表达量显著提高,在25 d时表达量又低于正常供磷水平。ClPht1;2基因具有PHT1基因家族特征结构,在杉木不同组织中均有表达,在杉木根中的表达受低磷胁迫诱导,在叶中的表达量受低磷胁迫诱导不明显,可能为杉木体内低亲和的磷转运蛋白,参与杉木地上部和根中磷的运输和分配。

Phosphate transporter 1(PHT1)is an important gene family that mediates the absorption,transport and distribution of phosphorus in plants.To understand the gene structure and function expression of Chinese fir phosphorus transporter,a phosphorus transporter gene ClPht1;2 was cloned from the third-generation of the Chinese fir clone 061,and the temporal and spatial expression of ClPht1;2 under different phosphorus stresses was analyzed.Based on the core sequence of ClPht1;2 obtained by transcriptome sequencing,the full length sequence of the target gene was cloned by the rapid amplification of cDNA ends technique(RACE)using the root cDNA from Chinese fir clone YANG061 as a template,the expression of ClPht1;2 in different tissues and the expression of ClPht1;2 in the roots of 3,10 and 25 days after phosphorus stress were detected by real-time fluorescent quantitative PCR.A Chinese fir PHT1 gene ClPht1;2(GeneBank accession number:MK450598)was cloned.The results of multiple sequence alignment of phosphorus transporter family gene in different plants showed that the amino acid sequence similarity between ClPht1;2 and PHT gene of Cryptomeria japonica was 93%,and that between ClPht1;2 and the PHT genes of Pinus massoniana,Camellia oleifera and Populus trichocarpa was above 72%.The length of the ClPht1;2 gene sequence was 1565 bp,coding for 511 amino acids.The theoretical molecular weight was 60.024 ku,which was hydrophobic protein with no signal peptide,and 42 phosphorylation sites.The protein consisted of 12 transmembrane structures.The polypeptide chain was composed ofα-helix(42.65%),irregular coil(42.11%)and extended chain(15.25%).ClPht1;2 was expressed in the roots,stems and leaves,with the highest expression in leaves and the lowest expression in roots.Compared with normal phosphorus supply,the expression of ClPht1;2 in roots increased significantly under low phosphorus stress from the 10 th day,and decreased to normal phosphorus supply level at the 25 th day after low phosphorus stress.The expression level decreased from the third day under non-phosphorus starvation,and increased significantly at the 10 th day,and the expression level was lower than the normal phosphorus supply level at the 25 th day.ClPht1;2 gene has the characteristic structure of PHT1 gene family.The expression of ClPht1;2 was observed in different tissues of Chinese fir.The expression in roots was induced by low phosphorus stress,but the induction was not significant in leaves.ClPht1;2 may be a low-affinity phosphate transporter,which is involved in the transportation and distribution of phosphorus in shoots and roots of Chinese fir.