Cloning And Functional Analysis Of Trehalose-6-phosphate Synthase Gene (MeTPS1-3) In Cassava

Trehalose synthase genes are another kind of genes closely related to stress resistance after the finding of glutamic acid, proline, mannitol, and betaine synthase genes in the study of plant stress resistance. Trehalose-6-phosphate synthase (TPS) gene is the key gene in the trehalose synthesis pathway. At present, most trehalose synthase genes were cloned from a variety of microorganisms and transformed into plants, and transgenic plants with enhanced drought resistance were obtained. Trehalose-6-phosphate synthase genes in higher plant were seldom cloned. Manihot esculenta Crantz has strong drought stress resistance, but there is no reports about its TPS genes. Therefore, Our study on MeTPSl-3 gene has important theoretical and application value.We firstly studied the differential expression of trehalose synthase gene (MeTPSl-3) in cassava cultivars SC124 and KU50 under different simulated drought stress by real-time fluorescence quantitative polymerase chain reaction. Results showed that MeTPS1-3 expression level did not change significantly in the SC124 roots, stems, mature leaves and immature leaves after 2 h drought treatment. In the roots of KU50, expression level increased about 12 times, a small increase was detected in stems, while the level in immature leaves remained unchanged. The expression level in the roots and young leaves rose about 30 and 4 times after 24 h drought stress, respectively, while a small increase in stems and a little decrease in the mature leaves were observed. The expression level in roots of KU50 rose about 14 times after 24 h drought treatment, while in KU50, a little drop was detected in stems, mature leaves and young leaves. These observations suggested that the MeTPS1-3 gene plays an important role in drought resistance of cassava.The plant expression vector pCAMBIA2300-MeTPS1-3 was constructed in order to further verify the function of MeTPSl-3 gene. The MeTPSl-3 gene of cassava were transferred successfully into N. benthamiana via agrobacterium mediated method. Four kanamycin-resistant plants were obtained. PCR analysis demonstrated that the exogenous MeTPS1-3 gene had been integrated into the genome of transformed plants. RT-PCR and quantitative PCR analysis showed that the MeTPS1-3 was steadily transcribed in the roots, stem and leaves of the transgenic plants. HPLC/ELSD determination results showed that the transgenic plants accumulated trehalose to a content of 0.125-1.348 mg/g (FW) in different organs of the transgenic plants.The water loss rate of leaves removed from the T1 and T2 generation of the No. 1 transgenic plants and unit water loss was less than that of the non-transgenic plants. Under the natural drought stress, the transgenic plants showed was significantly stronger resistance compared to the non-transgenic plants. These results suggested that MeTPS1-3 gene played an important role in drought stress resistance in cassava.