Cloning,Functional Study And Expression Analysis Of Phosphate Transporter Gene EcPHT From Eichhornia Crassipes

Eichhornia crassipes could absorb nitrogen and phosphorus efficiently and therefore it could grow rapidly in eutrophic waters as an invasive plant.Its madness destroys the ecological balance of the water body a nd causes great economic losses,but it could use to purify water through control the Eichhornia crassipes growth,so we study the Eichhornia crassipes phosphate transporter(EcPHT)which was helpful to reveal the molecular mechanism of Eichhornia crassipes absorption in eutrophic water and the molecular mechanism of Eichhornia crassipes rapid invasion,providing a theoretical basis for the rational development and utilization of Eichhornia crassipes.Phosphorus is an indispensable nutrient element in plant growth and development,and is one of the important components of plant nucleic,such as acid,ATP and phospholipid.Studying the EcPHT could provide a reference for efficient phosphorus uptake economic crops to absorb phosphorus efficiently,and could provide new genetic resources for genetic breeding of excellent crops.In this paper,EcPHT degenerate primers were designed according to the conserved sequence of high affinity phosphate transporter(PHT1)in over twenty kinds of plants,and 710 bp conserved fragment of EcPHT gene was obtained.Then though the RACE method,the 5' and 3' end' fragments of the EcPHT gene were clonded,and the full length of EcPHT gene sequence was obtained by splicing.The complete open reading frame of EcPHT gene was 1572 bp,it encoded a polypeptide of 523 amino acids.The EcPHT protein was predicted that the molecular weight was 57.009 kD and PI=8.95.It contained a signal peptide and 11 transmembrane regions,and was predicted that it was a stable,hydrophobin protein.The structure of EcPHT gene was highly similar to MFS transporter superfamily and sugar transporter-like.The amino acid sequence of this gene was more than 80% similar to the plant PHT1 gene of Elaeis guineensis,Dendrobium catenatum,Citrus trifoliata and Citrus clementinat.Through the yeast functional complementation test,it was verified that the EcPHT gene could complement the function of the phosphorus-deficient yeast PHO84 in 175 ?MP,indicating that the gene could transport the phosphorus ions.The yeast PHO84 grown at different pH values,the yeast grown best in pH=6,the yeast with phosphate transporter grown better under acidic conditions.The Agrobacterium tumefaciens EHA105 mediated EcPHT gene was transferred to tobacco by leaf disc transformation,and the growth of transgenic tobacco at different phosphorus concentrations was observed.The results showed that the number of root in transgenic tobacco was more than that of the wild type,which was significant in transgenic Zhongyan100 tobacco.The transgenic tobacco grew better than the wild type under 0.1 mM low phosphorus conditions.This paper analyzed the expression patterns of EcPHT gene in roots and leaves of Eichhornia crassipes and transgenic tobacco,and the expression patterns of Nicotiana tabacum phosphate transporter(NtPHT)and Nicotiana tabacum phosphate starvation response(NtPHR)in transgenic tobacco under different culture conditions using fluorescence quantitative PCR method.The results showed that the phosphate transporter gene expression level in Eichhornia crassipes root could be improved under low phosphorus stress,and the expression of EcPHT gene in the root of Eichhornia crassipes was significantly increased at 72 h under 0 mMP condition.The expression level of EcPHT gene in Eichhornia crassipes leaves was suppressed when Eichhornia crassipes was cultured under phosphorus deficiency and then transferred to 1 mMP.After 40 days of incubation in MS medium,the EcPHT gene could be obvious expressed in transgenic tobacco root and leaves,the NtPHT gene expression was significantly increased in the transgenic Zhongyan100 tobacco root and slightly decreased in transgenic Yk180 tobacco root,the expression of NtPHR gene in transgenic Zhongyan100 tobacco leaves and root could be slightly increased.Under 0.1 mMP phosphorus conditions,the NtPHT gene expression in the Zhongyan100 tobacco root was induced and was the highest level in transgenic YK180 tobacco.Under 2mMP phosphorus conditions,the EcPHT gene expression in transgenic Zhongyan100 tobacco root and leaves could be increased,but the expression level of NtPHR gene in transgenic Zhongyan100 tobacco root and leave was improved.The molybdenum blue colorimetric method was used to detect the inorganic phosphorus content of Eichhornia crassipes and transgenic tobacco under different culture conditions,and the total phosphorus content in transgenic tobacco at different growth time.On the first day,the content of inorganic phosphorus in the roots and leaves of Eichhornia crassipes was affected by the external phosphorus concentration.The inorganic phosphorus content of water hyacinth leaves increased with it growth under the condition of 1mMP.The inorganic phosphorus content in the tobacco roots were getting higher and higher with the external phosphorus concentration became higher,and the phosphorus content in transgenic tobacco leaves were getting higher and higher with the tobacco grown.After 30 and 40 days of incubation in MS medium,the phosphorus content in the transgenic tobacco leaves infected with Agrobacterium were higher than that in the roots.