Research On The Physiological Mechanism Of Salt Tolerance In Rosa Rugosa Thunb.and Mining Of Salt Tolerance Regulatory Transcription Factors

Rosa rugosa Thunb.belongs to the shrub of Rosa in Rosaceae,and can be divided into wild R.rugosa and cultivated R.rugosa.Cultivated R.rugosa are rich in rose essential oil,which is widely used in spice industry.However,due to long-term sweet soil cultivation and artificial selection,its salt tolerance has deteriorated significantly.Wild R.rugosa are naturally distributed in coastal beaches and have strong salt tolerance.Based on the above background,this study explored the physiological mechanism of salt tolerance of wild rose by paraffin section and non-invasive micro-measurement,screened the regulatory factors of salt tolerance by transcriptome sequencing,and preliminarily verified the regulatory mechanisms of two key transcription factors by heterologous expression of Arabidopsis thaliana.The main results are as follows:1.The morphological structure of leaves,stems,lignified taproots and white young roots of wild R.rugosa were observed by paraffin section.It was found that there was no specialized structure or tissue in wild R.rugosa,so it was excluded that wild R.rugosa depended on specialized tissues such as salt glands or salt vesicles to resist salt stress.The fluorescence detection of Na+ in the roots of salt-treated rose seedlings showed that Na+ accumulated in the root cells of wild R.rugosa,which was significantly higher than that of cultivated R.rugosa.It was speculated that the roots of wild R.rugosa had ion segregation.Further,the nondamaged Na+ flow in roots of wild R.rugosa was measured within 8 h under salt stress,and it was found that Na+ in roots of wild R.rugosa always showed a state of efflux,and the efflux flow rate gradually decreased with the extension of time,and the rate of single-assignment was significantly higher than that of cultivated R.rugosa.From the above results,it is preliminarily judged that compared with cultivated R.rugosa,wild R.rugosa store salt ions through ion compartmentalization of root cells to prevent ions from transferring to aboveground tissues,and at the same time,they have strong ion efflux ability to eliminate excessive Na+.2.Transcriptome sequencing was carried out on leaves and roots of wild R.rugosa treated with salt,and gene expression profiles were established and 17,253 differentially expressed genes were predicted,including 240 transcription factors.Six transcription factors RrLBD40,RrCOL1,RrGLK,RrC2H2,RrC3H and RrHRTL,which have the same up-and-down trend in roots and leaves,were selected as candidate genes.Candidate gene cloning analysis showed that these six transcription factors had DNA binding domains of their corresponding families,and subcellular localization experiments confirmed that they were all located in the nucleus.In the wild R.rugosa treated with 170 mM NaCl for 1 h,the expressions of RrLBD40 and RrCOL1 in leaves and roots increased,while the expressions of RrGLK,RrC2H2,RrC3H and RrHRTL in leaves and roots decreased.3.35S overexpression plants of RrLBD40 and RrCOL1 were obtained by constructing overexpression vectors of six genes and heterologous transformation in A.thaliana.It was found that the root system of transgenic A.thaliana with RrLBD40 was longer than that of wild type under salt stress.However,observing Na+fluorescence,it was found that transgenic A.thaliana did not show obvious difference with WT plants under the same concentration of salt stress.Transcriptome sequencing of transgenic A.thaliana showed that RrLBD40 may regulate ROS clearance and ion transport by interacting with auxin response protein gene RrSUAR and calcium-dependent cation channel protein gene RrADVC,and regulate salt tolerance of wild R.rugosa.RrCOL1 may regulate the salt stress response in series through the signal transduction pathway mediated by plant hormones.Therefore,RrLBD40 and RrCOL1 can be used as candidate genes to obtain transgenic roses by genetic engineering.