Function Analysis Of PsSUT1 And PsSUT2 Involved In Sucrose Transportation In The Peony

Peony(Paeonia suffruticosa)is a traditional Chinese famous flower,but planting peonies in pots will affect their growth and development,which greatly reduces their ornamental value and restricts the further development of the peony industry.Studies have shown that pot planting of peony can cause sucrose supply to be hindered during its growth and development,which affects its nutritional transport.The research team obtained and cloned the full-length c DNA of Ps SUT1 and Ps SUT2 genes related to sucrose transport of peony based on the previous research.In this study,protein structure analysis,subcellular localization,sucrose transport activity analysis of the two genes were carried out,and the transgenic Changes in physiological indexes,photosynthetic characteristics and morphological characteristics of Arabidopsis plants.Finally,the Ps SUT2 gene was heterologously transformed into petunia for functional verification.The results show that:Based on the results of the previous RACE of the research group,the sucrose transporter Ps SUT1(KC542395)and Ps SUT2(KC542396)genes were cloned from peony,respectively.The open reading frame of the Ps SUT1 gene is 1 557 bp,encoding 519 amino acids;the open reading frame of the Ps SUT2 gene is 1 803 bp,encoding 601 amino acids.A total of 6 conserved motifs were identified by analyzing the conserved motifs of the amino acid sequences encoded by the two genes,and the positions of the six conserved motifs were consistent.Motif 1 is a conserved domain of the MFS transporter family.Analysis of the tertiary structure of the peony SUTs protein by homology modeling found that the tertiary structure of the Ps SUT1 protein and the Ps SUT2 protein matched the same template and the structural similarity was extremely high,indicating that the peony SUT family protein was relatively conservative in the evolution process.Construct the GFP expression vector of Ps SUT1 and Ps SUT2 genes of peony,and inject it into tobacco leaves to make it transiently expressed in tobacco leaves.Observation by laser confocal microscopy revealed that the Ps SUT1 and Ps SUT2 proteins of Peony were localized in the plasma membrane.By constructing the yeast expression vectors of Peony Ps SUT1 and Ps SUT2 genes,they were transformed into yeast SUSY7/ura3 strains with sucrose absorption defects,so that they could be heterologously expressed in yeast.Both Ps SUT1 and Ps SUT2 genes showed complementary functions on SD sucrose selection medium,while the negative control empty p DR196 showed growth inhibition.The growth of the two gene expression vectors and the unloaded p DR196 on the SD glucose selection medium were the same, both were normal.It showed that both Ps SUT1 and Ps SUT2 genes of peony can encode membrane transporter with sucrose transporting activity.The Ps SUT1 and Ps SUT2 genes of Peony were heterologously expressed in Arabidopsis thaliana.After screening and identification,T1 generation Ps SUT1 and Ps SUT2 transgenic positive seedlings of three strains were obtained.Observing the growth of the T1 generation transgenic positive seedlings separately,it was found that the above-ground biomass of the Ts generation positive transgenic seedlings of Ps SUT1 and Ps SUT2 genes was significantly higher than that of wild-type Arabidopsis,including plant height,stem diameter and number of seed pods.Ps SUT2 gene T1 transgenic positive seedlings also appeared to bolting and flowering in advance.The photosynthetic fluorescence parameters of the transgenic plants were determined.By combining the rapid chlorophyll fluorescence induction kinetics curve and the "JIP-test" parameter analysis,it was found that Ps SUT1 transgenic plants compared with wild-type plants ABS/RC,DIo/RC,TRo/RC and The value of ?Do decreases,and the values of ETo/RC,?Po,?o,?Eo,PIabs,and PItotal increase.The value of the “JIP-test” parameter of Ps SUT2 transgenic plants is opposite to that of Ps SUT1 transgenic plants.This indicates that the heterologous expression of Ps SUT1 and Ps SUT2 genes in peony may cause different changes in plant leaf physiological state and photosynthetic apparatus.The soluble sugar test showed that among the three transgenic Arabidopsis leaves,the total soluble sugar content and sucrose content of the Peony Ps SUT1 transgenic Arabidopsis leaves decreased significantly,while the total soluble sugar content and sucrose content of the stems were both significant.Down.The content of sucrose in leaves of Ps SUT2 transgenic plants decreased significantly,the soluble sugar content of two lines did not change significantly,one line decreased significantly,while in the stem,soluble sugar content and sucrose content increased significantly.The sucrose tolerance experiment was carried out on the T1 generation transgenic positive seedlings of Ps SUT1 and Ps SUT2 genes respectively.The results showed that the transgenic positive seedlings and the wild type can grow normally on the normal concentration of sucrose(3%)MS medium,but the Ps SUT1 and Ps SUT2 gene transgenic positives The root system of the seedlings is more developed.On high-concentration sucrose(6% and 9%)MS medium,the growth of wild-type Arabidopsis thaliana is inhibited,and there is a phenomenon of short plants,weak root and leaf growth,and anthocyanin accumulation in the leaves.Both Ps SUT1 and Ps SUT2 transgenic plants grew normally and the root development was enhanced.This indicates that the Ps SUT1 and Ps SUT2 genes of Peony can effectively improve the efficiency of sucrose transport in plants,thereby promoting plant growth and development.In addition,the Ps SUT1 and Ps SUT2 genes of Peony can effectively alleviate the effects of high-concentration sucrose on plants.In order to further verify the effect of the Ps SUT2 gene on the flowering process of the plant,in this study,the peony Ps SUT2 gene was heterologously expressed in the ornamental plant Petunia hybrida.The results showed that the transgenic petunia flowered earlier than the wild type.The plant After 30 days of transplanting,the growth was more vigorous than the wild type.Heterologous expression of Ps SUT2 gene in Arabidopsis was similar.It is speculated that the Ps SUT2 gene not only promotes sucrose transport in plants,but also may participate in regulating the flowering time of plants.