Study On The Regulation Of RBOHA/RBOHD Gene Expression In Brassica Campestris L.under Abiotic Stress

Plant NADPH oxidase is also known as the respiratory burst oxidative homologous protein,which is a kind of cytoplasm of NADPH as an electron donor,the oxygen catalyzes the production of reactive oxygenase oxidase.As one of the main sources of"oxygen explosion",NADPH oxidase plays an important role in regulating plant growth and development,stress and cell signal transduction by regulating the production of reactive oxygen species.Several reports show that plant NADPH oxidase gene is a gene family,the different RBOH gene are different in tissue distribution and gene expression and perform different functions.In this report,using Brassica campestris seedlings Longyou 6 and Tianyou 2 as studied materials to investigate the pattern of RBOHA/RBOHD gene under different stresses and study the relationship between MAP kinase signal transduction pathway,NADPH oxidase and H₂O₂ production under low temperature and high salt treatment conditions in order to supplement the new theoretical basis for the mechanism of plant resistance to abiotic stress.The main results are as follows:1.The RBOHA/RBOHD gene of Brassica campestrisis expresses in roots,stems,leaves,hypocotyl with almost no tissue specificity.2.Brassica campestris seedlings Longyou 6 and Tianyou 2 are treated with low temperature?2??,high salt?200 mM?at different time and ABA,H₂O₂,PEG at different concentrations,the RBOHA/RBOHD gene expression is higher than the control.In addition,the expression of RBOHA/RBOHD gene in Longyou 6 is higher than that in Tianyou 2.The results show that RBOHA/RBOHD gene plays an important role during plant response to low temperature,high salt,ABA,H₂O₂ and drought stress.3.The results of prementment with NADPH oxidase inhibitor?DPI?,MPKK specific inhibitor?U0126?and H₂O₂ scavenger?DMTU?then treatment with low temperature or salt stress compare with those of low temperature or salt stress alone,the RBOHA/RBOHD gene expression is inhibited in Longyou 6 and Tianyou 2.The results show that there is a cross talk mechanism between transcription of these two genes and MPK cascade pathway and H₂O₂.4.The reactive oxygen species content,including H₂O₂,·OH and the activities of the antioxidant enzymes,including SOD,POD,CAT and APX,are studied in low temperature or salt stress,respectively.The results show that low temperature or salt stress leads to an increase of H₂O₂ and·OH concentration,followed by an increase of SOD,POD,CAT and APX activity.This shows that the low temperature or salt stress treatment at different time,resulting in differences in the content of reactive oxygen species in the plant,and ultimately affect the activity of antioxidase enzymes.5.After pretreatment with DPI,U0126 and DMTU,the reactive oxygen species contents?H₂O₂,·OH?and antioxidant enzymes activity?SOD,POD,CAT,APX?are significantly inhibited in Longyou 6 and Tianyou 2 compared with single low temperature and salt stress.The results show that reactive oxygen species induced by low temperature or salt stress are mainly derived from NADPH oxidase.Reactive oxygen and MPK cascade pathway are involved in the increase of antioxidant enzyme activity induced by low temperature and salt stress.The above data indicate that the RBOHA/RBOHD gene of Brassica campestris L.plays an important role in abiotic stress.Low temperature and high salt induce the expression of RBOHA/RBOHD gene,produce H₂O₂,activate MPK cascade system,and induce antioxidant defense System to remove the excess ROS,the activated MPK re-activates the expression of the RBOHA/RBOHD gene,which in turn increases the production of ROS.Therefore,there is a cross-talk mechanism between H₂O₂,NADPH oxidase and MPK cascade system,they work together to reduce the plant injury caused by stress.