Cloning And Functional Analysis Of Membrane-Vesicle Transport-related Gene RtSYP121 And RtVAMP1-3 From Reaumuria Trigyna

Reaumuria trigyna belongs to the genus Reaumuria Linn of the family Tamaricaceae.It is a strong xerophyte salt-secreting halophyte.It is mainly distributed in the arid desert areas of central and western Inner Mongolia,and has strong adaptability to saline-alkali and arid environments.Exploring the stress resistance mechanism of R.trigyna is of great significance to the development and utilization of this highly resistant native plant.In this thesis,based on the salt-tolerant transcriptome data of R.trigyna,two membrane vesicle transport-related genes,RtSYP121 and RtVAMP1-3,were screened and cloned,and their gene expression characteristics and subcellular localization were analyzed.The gene was transferred to Arabidopsis thaliana to verify its function under abiotic stress,laying a foundation for a deeper understanding of the regulatory functions and resistance mechanisms of RtSYP121 and RtVAMP1-3 genes in plant resistance to adversity stress.The main results are as follows:1.In this study,the RtSYP121 and RtVAMP1-3 genes were screened and cloned based on the salt-tolerant transcriptome data of R.trigyna.The ORF sizes are 888bp and 714bp,encoding 295 and 237 amino acids,respectively.Analysis of q RT-PCR results showed that RtSYP121 and RtVAMP1-3 can be expressed in the roots,stems and leaves of Reaumuria trigyna,which is the highest in the roots.It can be induced simultaneously by salt,drought,low temperature,and CdCl₂treatment.That it is on the cell membrane.2.We construct pPZP221-RtSYP121 and pPZP221-RtVAMP1-3 eukaryotic expression vectors and transform Arabidopsis thaliana to obtain stable gene strains.The wild-type(WT)and transgenic Arabidopsis were treated with salt,drought and CdCl₂stress.The seed germination rate,biomass,and chlorophyll content of the transgenic lines were better than those of WT;at the same time,the active oxygen content and oxidation of the transgenic lines The degree of damage was significantly lower than that of WT,but the activity of antioxidant enzymes(SOD,POD,CAT)and proline content were significantly higher than that of WT.The results of q RT-PCR showed that the stress-related genes AtSOD1,AtPOD1,AtAPX1,AtSOS1,AtNCED3,AtHMA2,AtHMA4,AtRD22 and AtRD29A could be induced to express in varying degrees.It indicates that RtSYP121 and RtVAMP1-3 genes may increase the resistance of transgenic Arabidopsis to adversity stress by increasing the antioxidant enzyme activity,osmotic regulation ability and the expression of stress-related genes in transgenic Arabidopsis.3.The ion content and flow rate of Arabidopsis thaliana under stress conditions were analyzed.The results showed that compared with WT,RtSYP121 transgenic Arabidopsis thaliana accumulated less Na⁺and more K⁺under salt stress,maintained a lower Na⁺/K⁺,and promoted Na⁺efflux;RtSYP121 transgenic Arabidopsis has higher K⁺and Ca²⁺content,lower Na⁺/K⁺and Cd²⁺influx rates under CdCl₂stress;these indicate that RtSYP121 regulates transgenic Arabidopsis ions The content and flow rate are kept low Na⁺/K⁺to enhance the tolerance of transgenic Arabidopsis to salt and CdCl₂stress.Compared with WT,RtVAMP1-3 transgenic lines have lower Na⁺content and K⁺efflux rate under salt stress,and higher Na⁺efflux rate,but there is no significant difference in Na⁺/K⁺;transgenic lines accumulated under drought stress More Ca²⁺,and a higher rate of Na⁺and K⁺efflux;these results indicate that RtVAMP1-3 mainly promotes Na⁺efflux,reduces Na⁺content,Atthe same time,it promotes the accumulation of Ca²⁺to enhance the tolerance of transgenic Arabidopsis to salt and drought stress.4.The cell viability of the roots of WT and transgenic Arabidopsis under CdCl₂stress was analyzed,and the results showed that compared with WT,the transgenic lines had stronger cell viability and stronger tolerance to cadmium stress.The structure changes of RtSYP121 transgenic lines and WT Golgi and chloroplasts under CdCl₂treatment were observed by transmission electron microscope.The results showed that,compared with WT,the Golgi flat capsules of RtSYP121 transgenic Arabidopsis thaliana were larger,and the ends appeared obviously larger in volume and quantity.There are more secretory vesicles;at the same time,the volume of starch granules in the chloroplast becomes smaller and the number decreases.Therefore,RtSYP121 may increase the plant's tolerance to heavy metal cadmium stress by increasing the activity of Arabidopsis vesicle transport and the disintegration of starch grains in chloroplasts under stress conditions.