Response Of Tea Plant Sulfate Transporter Genes To Selenium And Functional Analysis Of CsSULTR3.5

Selenium?Se?is an essential trace element for humans.Whether Se is rich in food is closely related to human health.Adverse effects would be caused by Se deficiency or excess.Most of Se in human diet is obtained from plants in a directly or indirectly way.In our daily diet,the level of Se intake is directly affected by the content of Se in edible parts of plants.Globally,the phenomenon of Se deficiency is very common.Tea is an ideal resource for Se supplement for its higher Se concentration as well as bioavailability.Further studies on the mechanism of Se absorption and transport in tea plant are of great significance for Se-enriched breeding and genetic improvement of tea plant.Eight cDNA sequences of CsSULTRs were obtained by RT-PCR on the basis of sequence resource from tea genome and transcriptome.Their tissue-specific expressions and responses to various Na₂SO₄or Na₂SeO₄ treatments were analyzed by quantitative real-time PCR.The preliminary functional analysis of CsSULTR3.5 has been carried out.The main results were summarized as follows:1.Bioinformatics analyses showed that all the CsSULTR sequences contains complete Open Reading Frame?ORF?,encoding 658,658,653,659,650,686,637,706 amino acid residues respectively.All of the CsSULTRs contained typical SLC26A/SulP transporter domain and STAS domain from sulfate transporters.According to the homology of amino acid sequences,CsSULTRs were named as CsSULTR1.1?GenBank accession No.KY963791?,CsSULTR1.2?GenBank accession No.KY963792?,CsSULTR2.1?GenBank accession No.KY977431?,CsSULTR3.1?GenBank accession No.KY963793?,CsSULTR3.2?GenBank accession No.KY977432?,CsSULTR3.3?GenBank accession No.MF596178?,CsSULTR3.5?GenBank accession No.MG792805?,CsSULTR4.1?GenBank accession No.KY963794?respectively.2.The expression patterns of CsSULTRs genes were varied under different Na₂SO₄ or Na₂SeO₄treatments.In annual tea plants,CsSULTR1.1 was induced in roots under S deficiency in a short time.CsSULTR1.2 has a specific response to Se treatments in roots.The expressions of CsSULTR2.1 and SULTR3s were induced by S deficiency as well as Se additional conditions.The tracscript level of CsSULTR4.1 was not affected by neither Na₂SO₄ nor Na₂SeO₄ treatments.3.The tissue-specific expression analyses revealed that CsSULTRs genes expressed in the organs of roots,stems,leaves,flowers and fruits with varied transcript levels.In annual tea plants,CsSULTR1.1and CsSULTR1.2 was abundantly expressed in roots,CsSULTR2.1 and SULTR3 were specifically expressed in stems,and CsSULTR4.1 was mainly expressed in stems and leaves.However,in 3 year-old tea plants,CsSULTR1.2 was primarily expressed in stems,CsSULTR2.1 had relatively high expression level in flowers,and CsSULTR4.1 was specifically expressed in reproductive organs.Furthermore,CsSULTR3.5 exhibited very high transcript abundance in all the detected tissues.4.Subcellular localization analysis showed that CsSULTR3.5 was located in plasma membrane,which was demonstrated by construction of transient expression vector CsSULTR3.5-GFP followed by transformation of rice protoplasts by PEG-mediated.The nucleotide sequence of CsSULTR3.5 promoter in length of 2 516 bp was cloned from tea plant.Through online prediction analysis of putative cis-acting elements,it was found that the promoter contained not only specific promoter structure,but also hormone response elements,light responsive elements and stress response elements,etc.It indicated that the expression of CsSULTR3.5 can be regulated by various factors.The analysis of tissue expression specificity of pCsSULTR3.5::GUS transgenic lines suggested that,the GUS activity could be detected in all organs except seeds as well as cotyledon.Moreover,the highest GUS activity was detected in vascular tissue.There is no significant difference in phenotypes between CsSULTR3.5over-expressed Arabidopsis and wild type under various Na₂SO₄ and Na₂SeO₄ treatment conditions.