Cloning And Expression Analysis Of BnMKK4Gene And Research On Separation And Activity Of BnMKK4Promoter In Brassica Campestris (Rape)

Plants are often exposed to environmental stresses throughout their life cycle，butthey deployed a variety of defense mechanisms to protect themselves from variousenvironmental stresses in the long-term evolutionary process. Mitogen-activatedprotein kinase (MAPK) pathway plays an important role in response to biotic andabiotic stresses in plants. The MAPK pathway exists in all eukaryotic cells. TheMAPK cascades are composed of MAPK, MAPKK and MAPKKK. The conversionand amplification of extracellular stimuli are performed through phosphorylation ofMAPKKK→MAPKK→MAPK in turn. In the signal transduction pathway, MAPKKsaccept and integrate the signal from MAPKKKs, and pass the information todownstream MAPKs. So the researches on MKKs from Brassica campestrisconducive to explore its signal transduction mechanisms in the process of resistanceto environment stress. The constitutive promoters are widely used in plant geneticengineering, but can’t effectively regulate the expression of target gene in time andspace aspects. Therefore, the study on inducible promoter is very important.In recent years, many MAPKKs have been isolated, but the research onMAPKKs in Brassica campestris is little. In this study, a novel group C MAPKKgene, BnMKK4, was isolated from Brassica campestris. The expression of BnMKK4and the activity of BnMKK4promoter were studied. The main results are as follows:（1）A novel MAPKK gene, BnMKK4, was isolated from Longyou6(GenBank ID:JF268686). Bioinformatic analysis reveals that the cDNA is1317bp in length withan open reading frame which encodes330amino acids. The molecular weight ofthe predicted protein is36.5kDa, with the isoelectric point (PI) being9.01. Thepredicted protein is a hydrophilic protein which contains several phosphorylation sites.The advanced structure of the predicted protein mainly contains alpha helix andrandom coil. A phylogenetic tree analysis indicates that BnMKK4belongs to group C.（2）BnMKK4mRNA was expressed in the stems, leaves and hypocotyls, and thetranscript level in the leaves was highest. The expressions of BnMKK4gene increasedin stems, leaves and hypocotyls of Longyou6under low temperature stress, while inTianyou2plants only the expression of leaves increased. For cold stress, theconcentration of BnMKK4mRNA in Longyou6was up-regulated, while the expression level of BnMKK4in Tianyou2increased first but decreased afterwardsduring the treatment. For salt treatment, the expression of BnMKK4in Longyou6andTianyou2increased first but then decreased when the concentration of NaCl wasincreased, but the concentration of BnMKK4mRNA in Longyou6decreased moreslowly. These results suggest that the BnMKK4gene is involved in response to coldstress and salt stress and that Longyou6is better.（3）The fusion protein pBI121-BnMKK4-GFP was expressed transiently in onionepidermis. The confocal microscopic examination showed that the BnMKK4: GFPfusion protein was targeted into the nuclear. So BnMKK4is localized in the nucleus.（4）A999bp fragment upstream of the start codon was isolated from Longyou6bygenome walking. Analysis of the BnMKK4promoter showed that the cold, droughtand ABA responsive elements were both observed. The results showed that theBnMKK4promoter may be induced by cold, drought and ABA stress.（5）The different length of BnMKK4promoter deletions were inserted intopCAMBIA1302to fuse with GFP gene. The transient expression of the GFP proteinin onion epidermal cells was observed using a confocal microscope. The resultsshowed that the functional region of the BnMKK4promoter could be located withinthe range of-416to-271bp upstream of ATG.（6）The promoter of BnMKK4gene was inserted into the vector pCAMBIA1302togenerate recombinant plasmid with green fluorescent protein (GFP). The promoteractivity of BnMKK4gene was studied by transient expression system of onion undertemperature and salt stress. The results clearly indicate that BnMKK4promoter couldbe induced by low temperature and NaCl.（7）For UV-B stress, the activities of CAT, GPOD and APX increased for two testvarieties within certain stress time, while the contents of MDA and UV-absorbingcompounds also increased and the content of chlorophyll decreased. But comparedwith Tianyou2, in Longyou6the activities of antioxidant enzymes were higher, thecontent of MDA was less, UV-absorbing compounds were more and the content ofchlorophyll decreased more slowly.