Regulation Of AtBRNO9 And GSNOR1 Gene By BR And NO Signaling Pathways

In this paper, we selected and used the Arabidopsis thaliana Material:wild-type Col-0 (Colombia-0), BR synthesis-deficient det2, wild type WS (WassileWSkija), BR-insensitive mutant signal bril-5. They were used to study that how the hormone brassinolide (BR) and NO signaling regulate the Ferritin gene (AtBRNO9) and S-nitrosoglutathione reductase 1(GSNOR1).The first experimental part is mainly about the regulation of AtBRNO9 and Fe2+ stress by the BR and NO signal. The first section is by using Real-time PCR to measure the Relative Expression Level of the Ferritin gene (AtBRNO9) with brassinosteroid (BR) and nitric oxide (NO) treated, cold and salt treatments in Arabidopsis thaliana living for 2 weeks; The second section is mainly by measuring Fe2+ stress physiological indicators such as Malondialdehyde, Fv/Fm, Hydrogen peroxide, Superoxide anion and other indicators between BR and NO treatments. We concluded that moderating the Fe2+ stress was affected by NO signaling pathway and BR signaling pathway. This part drew the following conclusions:1. The high Relative Expression Level of the Ferritin gene (AtBRNO9) was measured after Salt stress and cold stress treatments. So AtBRNO9 genes played an important role in salt stress and cold stress environment. Both NO and BR signals play an important role in the regulation of AtBRNO9 gene expression, and we estimate that BR signal may be transmitted through the NO signaling pathway. Fe2+ stress cause the increase of the Relative Electrical Conductive which means the membrane damage rate increases, and the decrease of Fv/Fm ratio which means that PSll has already been damage in iron stress and it causes the inhibition of photochemical electron transport, and the increase of the oxidative damage indexes (MDA, H2O2, and NBT stain O2-·content).2. BR plays a role in protection of the plasma membrane in iron stress. However, when removing the NO, plasma membrane is protected in iron stress. In the det2, NO has dual regulation in protecting the plasma membrane in iron stress. In the ion stress, the photochemical electron transfer is maybe negatively regulated by NO (reduced by 13.4%), but nothing to do with the BL signal. 3. Both BL and NO can protect against oxidative damage caused by iron stress, and Col-0 and det2 have the same performance:NO plays an important role in recovering oxidative damage caused by MDA, and oxidative damage caused by iron stress can not be recovered with exogenous BL or not. Both NO and BL play an important role in recovering oxidative damage causeed by H2O2, and BL can also work without NO. Both NO and BL play an important role in recovering oxidative damage causeed by O2-·and BL maybe work through the NO signaling.The second experimental part is mainly about constructing some subcellular localization, overexpression, RNAi and AmiRNA vector of S-nitrosoglutathione reductase 1 (GSNOR1) genes, and screening the Arabidopsis transgenic plants and expecting to find out the interaction between the BR and NO signaling. This part drew the following conclusions:1. The results of GSNOR1 protein bioinformatics preliminary analysis:full-length protein encoded by 379 amino acids, and the protein is highly rich in amino acids Ala (7.92%), Gly (10.55%), Val (10.82%); GSNOR1 proteins are hydrophobic regions; SOPMA software tested various types of secondary structure:a helix (25.07%), Extended strand (25.86%),βangle (9.5%), random coil (39.58%).2. We constructed some GSNOR1 vectors as follow:the overexpression vectors (GN1-forward-pEarlyGate101), subcellular localization vector (GN1-forward- pEarly Gate100), GSNOR1 gene RNA interference vector (GN1-reverse-pEarlyGate100, GN1-reverse-pMDC7-1), and GSNOR1 gene AmiRNA interference vector (GN1-AMI1-pEarlyGate100, GN1-AMI1-pMDC7, GN1-AMI2-pEarlyGate100, GN1-AMI2-pMDC7).3. All vectors of GSNOR1 had been infected to Col-0 and det2, and we gained the T1 transgenic plants, and screen of the homozygous plants is ongoing. We expect to gain those transgenic plants gene, and to observe the differences phenotype in gene expression and deletion plants, and then to measure a series of physiological indicators, and further to investgate the GSNOR1 protein function, and finally to investigate whether BR regulates the GSNOR1 gene which has a important role in NO metabolism, or not.