Cloning And Expression Analysis Of RBOHB Gene In Brassica Campestris L

When plants suffer biotic or abiotic stress, NADPH oxidase through producing a large number of signal molecular reactive oxygen species?ROS? in a short period of time, which regulates gene expression and cell metabolism, make the plants cope with adversity stress response in time and adapt to the changes of the environment. In this study, we use Longyou 6 and Tianyou 2 as materials, RBOHB as the research object, We did a preliminary study about the characteristics of the gene, biology information, transcription of the gene sequence of two kinds of Brassica campestris L. The main results are as follows: 1. We cloned a new RBOH family gene RBOHB from Brassica campestris L. Longyou 6. The full-length cDNA of RBOHB is 2694 bp, with open reading frame?ORF? 2541 bp, encoding a 846 amino acid protein. The predicting protein molecular weight MW is 96.7 kDa, and isoelectric point is 12.66. Senior predict protein structure show that RBOHB rich in alpha helix and irregular curl, the hydrophilic protein, no transmembrane helical structure. 2. The real-time fluorescent quantitative PCR?RT-PCR? results show that the RBOHB gene were expressed in the stem and leaf, hypocotyl in the two kinds of Brassica campestris L.Longyou 6 and Tianyou 2, no tissue specificity, and the expression amount in the leaf is highest. Low temperature, high salt, H₂O₂ stress can induce the two kinds of Brassica campestris L. RBOHB gene expression in different degrees, and Longyou 6 RBOHB transcription level was significantly higher than in Tianyou 2, which indicate RBOHB gene play a role in the proces against abiotic stresss in Longyou 6. MAPKK inhibitor U0126 pretreatment respectively 12 h then moved in low temperature, high salt, H₂O₂ stress, the results compared with in low temperature, high salt, H₂O₂ stress independently found that RBOHB gene transcription level were significantly decreased in two kinds of Brassica campestris L., but the amount of expression in Longyou 6 was higher than Tianyou 2, which showed that the restraining effect of MAPKK inhibitor U0126 to Tianyou 2 was stronger than Longyou 6. MAPKK inhibitor U0126 can also inhibit RBOHB gene expression, which showed the MAPKKK?MAPKK?MAP kinase of MAPK signal pathways have interaction with RBOHB gene transcription level, speculated that there are some sort of adjusting mechanism between them. 3. Two kinds of Brassica campestris L. were treated with different concentration?5mM?10 mM?15 mM?20 mM? of H₂O₂, APX and SOD activity presented rising first then downward trend with the increase of the H₂O₂ concentration, CAT activity and GPX had no significant changes.Chlorophyll content presented the downward trend with the increase of H₂O₂ concentration in Tianyou 2, which appearered rising first then downward trend in Longyou 6, when H₂O₂ concentration was greater than 10 mM, two kinds of Brassica campestris L. leaf chlorophyll content had the varying degree of decline, but Longyou 6 is still higher than in the Tianyou 2. Malondialdehyde?MDA? content of Tianyou 2 is always higher than Longyou 6. The content of free proline?Pro? have obvious downward trend with the increase of H₂O₂ concentration, but the accumulation in Longyou 6 were significantly higher than Tianyou 2. From the results of the physiological indexes changes, we found that the resistance of Longyou 6 is stronger than Tianyou 2. NADPH oxidase activity rise on different levels, and pretreatment MAPKK inhibitors then H₂O₂ treatment, the NADPH oxidase activity of Longyou 6 and Tianyou 2 to compare with H₂O₂ treament alone are reduced.The result showed U0126 has inhibitory effect to the NADPH oxidase activity. This results that were compared with two kinds of Brassica campestris L. RBOHB gene transcription level of real-time fluorescent quantitative PCR results after H₂O₂ stress and MAPKK inhibitors pretreatment adversity stress again are consistent.