| Chinese fir(Cunninghamia lanceolate)is a unique species of coniferous fast-growing timber,whose growth and development are affected by nutrient phosphorus.However,due to the strong chemical fixation of phosphorus in the southern acidic soil,the soil available phosphorus is extremely lacking and the tree growth is inhibited.For a long time,the cultivation method of multi generation continuous cropping has aggravated the deficiency of soil phosphorus nutrition in the Chinese fir forest land,Therefore,it is very urgent to study and select the effective utilization of phosphorus in Chinese fir.Although in recent years,a great deal of research has been done on the use of phosphorus in china,obtaining some research results,but most of the current study focused on Chinese fir root system configuration,physiological response,maintenance of soil fertility and forest nutrient cycling,mainly through the determination of physiological and biochemical processes of Chinese fir.Due to the restriction of the sequence information of the whole genome and transcriptome,there is still a lack of studies on the molecular mechanism of the effective utilization of phosphorus in Chinese fir.In particular,there are few reports on the cloning and functional analysis of the phosphate transporter gene of Chinese fir,and the molecular mechanism of the regulation and control of phosphorus in the soil is not clear.Plants to absorb soil inorganic phosphorus is mainly dependent on the phosphate transporter gene Pht to achieve,to explore the structure and function of Chinese fir phosphate transporter key genes,reveal the molecular mechanism of Chinese fir phosphorus nutrient absorption and transport,have an important practical significance for the selection of high phosphorus utilization efficiency of Chinese fir seed and the improvement of the utilization efficiency of P in soil.In view of this,this paper used the 4,25,and 32 Chinese fir selected by the previous study of the mentor group as the experimental materials,RACE and fluorescence quantitative PCR were used to carry out the full length cloning,subcellular localization,spatio temporal expression analysis under low phosphorus stress on the high affinity phosphate transporter gene ClPht1;1.By using transcriptome sequencing,the transcriptome database of roots,stems and leaves of 32 clones of Chinese fir was established;the genes involved in phosphorus transport in the transcriptome were screened and their functions were annotated.2 genes c200084_g1 and c221441_g1 related to the transport of phosphorus in Chinese fir were cloned and analyzed in time and space under low phosphorus stress.The main results are as follows:1.Using RACE technology to obtain a Chinese fir phosphorus transporter Phtl gene,named ClPht1;1(GenBank accession number:KX302006).The coding sequence of the gene was 1967 bp,encoded a protein of 545aa,whose atomic composition was C275OH4198N698O751S28,The molecular weight was 59.95kDa,the isoelectric point was 9.04,and the instability coefficient was about 33.44.The fat coefficient was 82.57,and the total average hydrophilicity was about 0.226,which was judged to be hydrophobic protein.ClPht1;1 encoded protein had a typical structure of Phtl gene family,and was highly homologous with Japanese cedar phosphate transporter gene.Subcellular localization was present in the cytoplasm,and was distributed in Golgi and endoplasmic reticulum,which may be excluded from the nucleus,cell membrane and chloroplast.2.ClPht1;1 gene and Phtl family gene expression characteristics were same,mainly expressed in the root of Chinese fir,the expression in the leaf was low;There was a significant difference in the expression of different phosphorus use efficiency in Chinese fir families,at the same time ClPht1;1 gene and Phtl family gene expression characteristics were same,mainlgene expression induced by low phosphorus stress,phosphorus deficiency stress ClPht1;1 gene expression was significantly increased,the expression of ClPht1;1 gene was significantly decreased after phosphorus recovery,and the higher the degree of stress,the more significant the change of expression.3?Through the sequencing of the transcriptome data to remove impurities,76,829,210,82,165,990 and 77,534,078 Clean reads were obtained from the roots,stems and leaves of Chinese fir.A total of 413,806 Unigenes were obtained after reassembly,with an average length of 520bp.A total of 109,596 fir Unigenes were annotated in the NR database,75%of the mapping sequences had strong homology,17.8%of the Unigenes had more than 80%similarity;Based on the analysis of species distribution,Chinese fir Unigenes and high gymnosperm spruce and the Amborella trichopoda sequences were 7.5%and 2.9%of the matching degree.A total of 148,466 Unigenes were annotated in the GO database,where 727,287 of the predicted genes had at least one GO function annotation information;A total of 92,001 Unigenes in the KOG database was with specific protein function definition;A total of 57,042 Unigenes in the KEGG database had a meaningful match,at the same time,there were 132 KEGG metabolic pathways.A total of 49 genes were identified with the function of inorganic phosphate transport through the annotation of gene function in the Unigene database,and 25 of these genes were annotated to participate in the activity of inorganic phosphate transmembrane transport.4?The c200084_g1 and c221441_g1 two genes were cloned to obtain the full-length sequences of 1634bp and 1753bp,composed of 386 and 487 amino acids,respectively.The physical and chemical properties of the protein showed that the molecular weights of the two genes were 43.49 kDa and 53.06 kDa,and the isoelectric point was about 5.60 and 5.27,respectively.c200084_g1 was an unstable and hydrophilic protein,c221441_g1 was a stable and hydrophilic protein.Two genes encode proteins that had no signal peptides and were not secreted proteins.The c200084_g1 gene encoding protein had a transmembrane region,and the proportion of irregular Loop ring in the two class structure was up to 50.26%,the c221441_g1 encoding protein had two transmembrane regions,and the proportion of alpha helix in the two class structure was the largest of 49.9%.The BLAST results showed that the c200084_g1 gene of Chinese fir was more than 80%of the homology with other plant genes,and the consistency of the coding sequence was 90.96%.The BLAST results showed that the homology of c221441_g1 gene was low,mostly in about 40%,sequence alignment had a consistency of 66.33%.5?The expression of c200084_g1 gene was higher in the root system of Chinese fir,and there was no significant difference in the expression of root?stem and leaf in different families,and the expression was not induced by low phosphorus stress.The expression of c221441_g1 gene in roots was much higher than that in stems and leaves.With the increase of phosphorus utilization efficiency of different families,the expression of the gene was increased,and the expression of the gene was induced by low phosphorus.We hypothesized that c200084_g1 and c221441_g1 may be involved in the metabolism of organophosphorus compounds in plants,the c200084_g1 gene may have the function of carrying and transporting organic phosphorus in some plant cell walls,the c221441_g1 gene may have the function of organic phosphorus transport in the process of dephosphorylation in plants,specific functions also need further research. |
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