Construction Of Overexpression Vectors Of Tae-pre-miR9677 And Its Short Tandem Target Mimic And Wheat Transformation

Micro RNAs play an important role in regulating plant growth, development and adversity stress response.In recent years, Micro RNAs identification and functional research has became a hot spot in plants research. However, wheat, as an important food crop, lags far behind other plants in Micro RNAs researches, this situation need to be overcome. To reveal the functions of newly identified Micro RNAs Tae-miR9677 that highly expressed in wheat panicle related, plant expression vector of Tae-pre-miR9677 and Tae-miR9677 STTM were constructed which were controled by Ubiqutin promoter, and immature or mature embryo callus of spring wheat cultivar Mianyang 19 were used as metarials to make wheat transfermation in this study. The main results were as follows:(1) Development of Tae-miR9677 STTM expression vector and wheat transformationFirstly, Tae-miR9677 STTM sequence was designed and artificially synthesized, and expression vector of pCAMBIA3301-Tae-miR9677 STTM was constructed. This expression vector was controled by Ubiqutin promoter and Bar gene was controled by CAMV35 S promoter. Then, 3683 embryos of wheat cultivar Mianyang 19 were used as receptor to transform the pCAMBIA3301-Tae-miR9677 STTM by biolistic particle of BIO-RAD corporation. Forty-two regenerated plants with resistance to Phosphinothricin were obtained after cultureing on the selection medium with Phosphinothricin. 8 transgenic positive plants with Bar and Tae-miR9677 STTM were identified by PCR assay in the T0 generation, with the transformation frequency of 0.21%. Totally, 27 seeds of T0 generation were obtained. All these seeds were germinated and planted in green house, and 27 T1 plants were identified by PCR as well as Basta test. Finally, 3 transgenic positive T1 plants were obtained.(2) Development of Tae-pre-miRNA 9677 expression vector and wheat transformationFirstly, pre-Tae-miR9677 STTM sequence was designed and artificially synthesized,and expression vector of pCAMBIA3301-pre-Tae-miR9677 was constructed. This expression vector made the tae-miR9677 STTM under control of Ubiqutin promoter and Bar gene under control of CAMV35 S promoter. Then, Agrobacterium that contain the Tae-pre-miR9677 expression vector infect wheat, 1300 wheat mature embryos were infected.7 regenerated plants with resistance to Phosphinothricin were obtained after cultureing on the selection medium with Phosphinothricin. 1 transgenic positive plants with Bar and pre-Tae-miR9677 were identified by PCR assay in the T0 generation. Basta daub in the T0 generation plants to get 1 positive plants. Finally, 1 transgenic positive T0 plants were obtained.