Molecular Cloning And Expression Analysis Of CsPT4 And CsPT1 Gene From Tea Plant(Camellia Sinensis),Subcellular Localization And Function Analysis Of CsPT4

Phosphorus is one of the most important mineral elements in the life process of plants.Not only the composition of important molecules such as ATP,nucleic acid and phospholipids also play a key role in energy transfer,metabolic regulation and protein activation.Although many soils are rich in phosphorus,in the soil,the free inorganic orthophosphate concentration directly available as plants are very low,usually 1 to 10 μmol/L.The low availability of phosphorus in the soil is due to the positive charge of the orthophosphate leading to the rapid chelation of the cations,thus allowing the soil inorganic phosphorus to be highly fixed.Tea plants（Camellia sinensis（L.）O.Kuntze）is mainly grown in acidic soil,and its buds are used to produce the world’s three non-alcoholic drinks one of the raw materials of tea.In order to adapt to the lack of available phosphorus in acidic soils,tea plants formed a set of mechanism of low phosphorus tolerance in the process of long-term evolution.From the aspects of root structure,improvement of rhizosphere soil environment and expression of phosphate transporter proteins（Phts）Adjust to better adapt to low phosphorus environment.It is very important to understand the molecular mechanism of low phosphorus tolerance and to clone the related genes for the cultivation of fine varieties with low phosphorus tolerance and to improve the quality of tea.To date,most of the plant phosphorus transporters cloned and identified belonged to Phtl.Plants in order to adapt to this low-phosphorus environment has evolved a set of mechanisms,including changes in root structure,rhizosphere soil improvement,organic acid secretion,phosphorus transporter expression.It has become a hot research topic by cultivating the high efficient utilization and utilization of gene resources and cultivating the efficient utilization of phosphorus.Studies have shown that plants mainly absorb the available phosphorus in the soil by expressing a variety of phosphorus transporters on the cell membrane.The In this study,two high affinity phosphorus transporter gene CsPht1;4（CsPT4）/CsPht1;1（CsPT1）were cloned from tea plant by RACE cloning technique.The results were as follows:（1）In this study,the full length cDNA of the CsPT4 gene of the tea plant phosphorus transporter was obtained by homologous cloning,Camellia sinensis cv.Longjingchangye as the experimental material for the first time（GenBank accession number:KY132100）.The gene was 1642 bp in length and 1620 bp in open reading frame（ORF）,encoding 539 amino acids.Bioinformatics analysis showed that the molecular weight of CsPT4 gene was 59.12 KD,the theoretical isoelectric point（pI）was 8.51,and the typical Phtl family was characterized by "6-hydrophilic-6" transmembrane structure.Fluorescence quantitative PCR showed that CsPT4 was expressed in roots,stems,leaves and old leaves of normal tea plants,and the expression level of CsPT4 was higher in the old leaves and lowest in the roots.The expression level of CsPT4 in roots and leaves increased first and then decreased after treatment with low phosphorus（1 μM）.The expression of CsPT4 in root was higher than that in leaves at 48 h.After treatment with phosphorus deficiency（0 μM）,the expression level of CsPT4 uptake in roots and leaves increased with the control,and reached the maximum at 72 h and 48 h,respectively.（2）In this study,full-length cDNA of CsPTl gene of tea plant phosphorus transporter was obtained by RT-PCR,Camellia sinensis cv.Longjingchangye as the experimental material.The full length cDNA（GenBank accession number:KY886916）was obtained.The gene was 1687 bp in length and 1626 bp in open reading frame（ORF）,encoding 541 amino acids.The molecular weight was 59606.23（Da）and the theoretical isoelectric point was 8.79.The homology of the amino acid sequence was similar to that of Camellia oleifera（AFU07481.1）,Sesame（XP₀1073524.1）,Walnut（XP₀18827352.1）,Jatropha curcas（XP₀12085328）,Tobacco（XP₀19253650.1）Is 99%,87%,85%,85%,85%.HMMTOP predicted the topological structure of CsPTl.The results showed that CsPTl had 12 transmembrane domains and the conserved sequence GGDYPLSATIMS was located in the fourth transmembrane domain.The predicted results were consistent with the reported Phtl transmembrane domain.The expression level of CsPTl was higher than that of control at all time points.（3）The fusion expression vector of green fluorescent protein（GFP）and target gene was constructed by transient transfer of gene gun into the onion epidermis.The expression of CsPT4 subcellular localization was analyzed by confocal microscopy.Green fluorescence appears on the plasma membrane,and the study shows that the functional location of the CsPT4 gene expression protein is on the plasma membrane.The results showed that CsPT4 could complement the yeast mutant with phosphorus transport function and promote the uptake of phosphorus by yeast mutant under low phosphorus condition.Using the radioisotope 32P,the Km value of CsPT4 protein was 35.3 μM in yeast,which belongs to high affinity phosphorus transporter.