| Sweetpotato is one of the seven important food crops in the world.It has been widely promoted in many areas due to its low production cost,large biological output,and high availability.Sweetpotato is mostly grown in poor land,and the impact of environment to its biological yield is very important.Therefore,it is of great significance to study the structure and function of sweet potato related stress resistance gene.As a plant osmotic adjustment substance,trehalose can replace the hydroxyl groups of water molecules and biofilms such as biofilms and proteins when the plants are subjected to abiotic stress such as drought,high salt and high temperature,and maintain the stability of plant cell membrane structure and function.Trehalose-6-phosphate synthase(TPS)is a key enzyme in the unique synthetic pathway of plant trehalose and has an irreplaceable role.Many studies have shown that TPS can participate in plant stress response.In this study,a Unigene sequence highly homologous to At TPS1 was obtained from the sweetpotato transcriptome database under drought conditions.Specific primers were designed and Ib TPS1 was successfully cloned.The sequence characteristics and expression patterns were analyzed.The N-terminal 2-88 amino acid was truncated and modified,and the yeast expression vector was constructed to identify TPS activity and yeast resistance.The plant expression vector was constructed for subcellular localization and transgenic sweet potato resistance identification,and the mechanism of action also was studied.The specific results are as follows:(1)Ib TPS1 was successfully isolated from sweetpotato by RT-PCR.The full length of the sequence was 2811 bp,encoding 936 amino acids.It included two conserved domains: GT1-TPS and HAD-TPP.Its secondary structure is dominated by random coils and ?-helices,without ?-sheets.It is no signal peptide present and most likely located in the cytoplasm.It is also an unstable hydrophilic protein;(2)Analysis of tissue-specific expression patterns indicated that Ib TPS1 was expressed in different organs of sweet potato.The protein encoded by Ib TPS1 was the highest in roots and leaves,and the weakest in petiole and fibrous roots.the up-regulation of Ib TPS1 could be induced under high-permeability,high-salt and high temperature stress conditions.The subcellular localization of tobacco showed that the protein encoded by Ib TPS1 was not in the nucleus and chloroplast,and it was the most likely in the cytoplasm;(3)A modified sequence ?NIb TPS1 with a 2-88 amino acid cut-off at the N-terminus was successfully obtained.The Ib TPS1-p YX212 and ?NIb TPS1-p YX212 yeast expression vectors were successfully constructed.Yeast heterologous complementation experiments demonstrated that the Ib TPS1-encoded proteins all have TPS enzymatic activity.The ?NIb TPS1 transgenic yeast showed obvious growth advantage under high salt,hyperosmotic and oxidative stress conditions,which proved that the N-terminal redundant sequence inhibited the activity of Ib TPS1 enzyme.Expression of ?NIb TPS1 in transgenic yeast increases yeast resistance;(4)The plant expression vector ?NIb TPS1-p GWB5 was successfully constructed by Gatewy technology.After the plant expression vector was transformed into Agrobacterium,?NIb TPS1 transgenic sweet potato strains were successfully obtained by sweetpotato genetic transformation system;(5)Three ?NIb TPS1 transgenic sweet potato plants(abbreviated as ?T)?T-4,?T-7 and ?T-9 were selected for the study of gene stress resistance.The results of leaf disc stress test showed that under the conditions of high salt,hypertonic and oxidative stress,the color of wild type leaves was obviously yellow,the edge was burnt,and the percentage of chlorophyll content decreased significantly.The percentage reduction in chlorophyll content is as high as 1.5-6 times than the transgenic sweet potato.The above results demonstrate that the expression of ?NIb TPS1 in transgenic sweet potato improves sweet potato resistance. |
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