Functional Research Of CsSULTR1;1 And CsSULTR1;2 In Selenium Absorption Of Tea Plant(Camellia Sinensis)

Selenium is an essential nutrient for human body as well as a beneficial element for plants.Selenate is the main form of selenium that can be utilized by plants in the soil.Studies have shown that plants mainly absorb selenate through sulfate transporters.SULTR1;1 and SULTR1;2 belonging to the first subfamily are considered to be candidate genes involved in plant selenium absorption.Our research team had cloned 8 sulfate transporter genes in tea plant.Preliminary study found that CsSULTR1;1 and CsSULTR1;2 may play an important role in the processes of selenium absorption and transportation in tea plant.In order to further clarify the function of CsSULTR1;1 and CsSULTR1;2 in the process of selenium absorption and provide a theoretical basis for selenium nutritional regulation of tea plant.In this thesis,we employed gene cloning,biological information analysis,subcellular localization,transgenic Arabidopsis and qRT-PCR to study the expressing characterization and selenium and sulfur?Se,S?response mechanism of CsSULTR1;1 and CsSULTR1;2.The main results are as follows:1.The response of CsSULTR1;1 and CsSULTR1;2 to Se and S was clarified.Both S and Se⁶⁺adding,CsSULTR1;1 did not show a special response,but CsSULTR1;1 and CsSULTR1;2 showed significant up-regulated expression in the absence of S even with Se⁶⁺adding,indicating that the upregulation of CsSULTR1;1 may be induced by S deficiency.Under the treatment of S and Se⁶⁺adding,the expression of CsSULTR1;2 was obviously induced.Combined with the expression analyses under the condition of S deficiency,it can be found that CsSULTR1;2 has a specific response to higher concentrations of Se⁶⁺regardless of the presence or absence of S.Adding different concentrations of Se⁴⁺in S treatment,CsSULTR1;1 showed significant down-regulation at each time point,indicating that Se⁴⁺significantly inhibited the expression of CsSULTR1;1.Either under the treatment of 0.6 mg/kg Se⁴⁺and 3 mg/kg Se⁴⁺,transcription level of CsSULTR1;2 significantly increased on the third day,and then returned to normal levels,indicating that CsSULTR1;2 may also have a specific response to Se⁴⁺.In a word,regardless of the presence or absence of S,CsSULTR1;2 can significantly respond to the stimulation of selenium treatment,indicating that CsSULTR1;2 may play a major role in the selenium absorption process of tea plant.2.The response of CsSULTR1;1 and CsSULTR1;2 promoters to Se and S was studied.A promoter fragment about 2000bp in length was cloned.The promoters of CsSULTR1;1 and CsSULTR1;2 contain various hormone response elements and environmental stress response elements related to plant growth and development.GUS staining results of different tissues showed that the promoter of CsSULTR1;1 is mainly expressed in the roots and cotyledon tips of transgenic Arabidopsis.The promoter of CsSULTR1;2 was expressed in vascular bundle tissues such as roots,stems,petioles and leaf veins,as well as stigmas and pods of flowers.GUS staining results under different Se and S treatments showed that lack of S would significantly induce up-regulated expression of CsSULTR1;1 and CsSULTR1;2promoters.Selenate can significantly induce the up-regulated expression of the two target gene promoters in the presence or absence of S.3.The full-length CDS sequences of CsSULTR1;1 and CsSULTR1;2 were cloned,and their functions were deeply analyzed.The tissue specific expression results in different periods showed that CsSULTR1;1 was highly expressed in roots,while CsSULTR1;2 was highly expressed in stems.The gene expression of CsSULTR1;1 and CsSULTR1;2 in tea growing period in spring was higher than that in winter dormancy period.The expression level of CsSULTR1;1 in the growing period showed that the mature leaves were higher than the roots,while in the dormant period it was just the opposite.The expression level of CsSULTR1;2 in stems is much higher than that in roots during the growth and dormant periods.The subcellular localization in tobacco mesophyll cells and rice protoplasts showed that CsSULTR1;1 and CsSULTR1;2 were located in the cell membrane and cytoplasm.After 7 days of culture in a medium containing 0?mol Se6+,0.5?mol Se6+,5?mol Se6+,and 50?mol Se6+respectively,the root length of the two overexpression lines of CsSULTR1;1 was longer than that of the wild type and reached a significant or extremely significant level.However,there was no difference in root length between the overexpressed strain and the wild-type strain treated with 100?mol Se⁶⁺.An overexpression line of CsSULTR1;2 was consistent with the results of CsSULTR1;1 in root length.