The Construction Of PBI121-TPAS IhpRNA Vector Of A Rice Tn Pong And Agrobacterium-mediated Transformation By Regeneration Of Embryonic Callus

Mobile elements are genetic elements that can move, and sometimes spread within their host genomes, meanwhile, it is an important part of a highly repetitive in the genome. In recent years, people have isolated a class of very active MITEs transposon, namely mPing. The research of mPing will help us understand the origin and transposition mechanism of MITEs, however, mPing itself can not encode transposase enzyme but by the relevant transposon Ping and Pong.In order to further study the role of transposon,we constructed pBI121-TPAS ihpRNA vector using Agrobacterium-mediated genetic transformation method to obtain plant regeneration,and through the phenotype observation of transgenic plants and molecular testing,we discussed the role of the transposon in plant growth and development. According to Pong base sequence(NCBI:BK000586),primers were designed using PCR amplification method,and we amplified the two gene fragments using rice toal DNA as template,namely TPAS-1 and TPAS-2 which were 222bp fragment length. Identification of target sequences,we connected fragment 1 to pKANANIBAL vector, then connected fragment 2 to pSP72 vector. Even recombinant vector pSP72 of fragment 2 was cut , which was fragment 3,and connected it to recombinant vector pKANANIBAL of fragment 1. Finally, the recombinant vector pKANANIBAL were digested and we obtained the fragment of Pong containing the intron, finally we connected it to the expression vector pBI121 to construct the rice expression vector pBI121-TPAS ihpRNA.The song-qian and 124 of mature embryos callus of rice as receptor material,we operated genetic transformation of the constructed transformation vector Pong using Agrobacterium-mediated transformation technology to obtain transgenic plants. In this study,we optimized genetic transformation of rice and confirmed that the concentration of bacteria infection was OD600=0.2, infection time 3min, co-culture 3d, pre-culture 5-7d, kana 35 mg/L.In this study,we totally obtained 16 strains of transgenic plants,of which three were transgenic positive 124 varieties, and PCR testing had confirmed that the foreign gene had been integrated into the rice genome. From the phenotypic point of view,transgenic plants were small,less tillering,early flowering and low seed. Transgenic plants which were after screening would further define the relationship between the activity of transposons in rice and apparent genetic variation to provide some research material.