Study On The High Levels Of Gaba Accumulation In Transgenic Rice Grains

In order to increase y-Aminobutyric acid(GABA)contents in rice grains,we constructed two overexpression vector respectively enhanced the expression of OsGADl and OsGAD2 which are the key GABA synthases,and two RNA interference(RNAi)vector respectively suppressed the expression of GAB A transaminase 1(OsGABA-T1)gene and GABA transaminase 2(OsGABA-T2),two key enzymes that converts GABA to succinate semialdehyde(SSA).The recombinant vectors were transformed into the callus of high yield japonica cv.'Ningjing 1' by Agrobacterium-mediated method.The T-DNA region for OsGABA-T1 or OsGABA-T2 RNA interference in regenerating rice plants was integrated with rice genome in T0 generation transgenic plants through PCR analysis of hygromycin(Hyg)selection marker gene and target fragment contained target DNA and vector sequences.The positive T0 generation plants were selected based on the PCR detection and sown in soil to obtain the T1 generation.We found that the level of OsGAD1 gene did not increase in the positive T1 plants of OsGAD1OE by qRT-PCR and the GABA contents were not higher than control.While,the level of OsGAD2 gene is higher in the positive T1 plants of OsGAD2OE than control,the GABA content did not increase also.So,there was no subsequent cultivation and study.We selected 6 stains of stable expression of T2 generation of OsGABA-TIRNAi transgenic plants for subsequent validation.The qRT-PCR for OsGABA-T gene expression in leaves and seeds of OsGABA-T1RNAi lines showed that there were much lower level of OsGABA-T1 and OsGABA-T2 transcript abundance in RNAi transgenic lines compared with those for Ningjingl.And compared with control,the OsGABA-T 1 protein was much lower in leaves of T2 generation of OsGABA-T1RNAi,T2-1 and T2-4.The GABA contents of brown rice in T3 generation of OsGABA-T1RNAi lines were 13 times higher than Ningjingl,also in polished rice,while the other main amino acid did not change much.We determined the GABA contents in four months of post-harvest storage brown rice as well as polished rice,and found that the level of GABA is still higher in transgenic rice,while we did not find any GABA in Ningjingl.We expanded the planting of T3 generation of OsGABA-T1RNAi plants,and then choosed 30 stains to investigate agronomic traits in each family.The result showed that the main agronomic traits of Ningjingl were better than transgenic lines which had shorter plant height,lower seed-setting rate and decreased 1000-grain weight.The RNAi fragment of OsGABA-T1 and OsGABA-T2 was respectively cloned into a double expression vector pSB130.We have obtained the seeds of T2 generation plants,in which the level of GABA is higher than Ningjing1.However,we failed to obtain the positive transgentic plants which only contain the target fragment but no Hyg selection marker gene.In future study,we need to screen for the positive transgentic plants without marker gene via PCR,Southern blot and other assays.In summary,through the study in this paper we can breed a rice variety which could contribute to reduce blood pressure by the enrichment of GABA content in rice seeds though the RNA interference of OsGABA-T gene.The nagetive effects of transgenic plants can be improved by replacing the promoter in the future study.