Cloning And Expression Analysis Of BnMPK6Gene In Brassica Napus (Rape) And Effect Of MAPKK Inhibitors On The Antioxidant Enzyme Activity Under Low Temperature Stress

Brassica napus (rape) is one of the world’s major oil crops, and the largest canolacrop in our country. The new rapeseed varieties Gansu oil has a high strength coldresistance, outstanding yield and wide adaptability, it can be adapted to extreme lowtemperature of-20to-30℃in arid region of northern China cultivation cultivation incold areas, is currently the only one can in Gansu Province, north-central Hexiregional security wintering of winter oilseed rape varieties. Plant growth, fixed,forcing it had suffered a variety of biotic and abiotic stress.MAP kinases(mitogen-activated protein kinase) is cascade channels important signal transductionpathway in plants, can be linked to cell membrane receptors and cell responses of thedifferent, endogenous, exogenous signaling plays an important role in the process.By on rape MAP Kinase Gene BnMPK6and promoter of clone, bioinformaticsanalysis, expression analysis and MAPKK single-minded inhibitor U0126under lowtemperature stress comparison longyou6and tianyou2, rape physiological index ofeffect research, for rape cold breeding and the resources identification and filterprovides must of foundation information, but also provides a theoretical basis forfurther exploration of plant cold hardiness, as well as to elucidate the function of theMAP kinase cascades in the process of plant stress.The results are as following:1. A new MAPK cDNA BnMPK6was isolated from Brassica napus. The clonedfull-length cDNA was1521bp, contained an1185bp ORF, a5’-untranslated region(UTR) about81bp, and a3’-untranslate region (UTR) about255bp. The nucleotidesequence of BnMPK6was85.4%identical with that of AtMPK6, so named itBnMPK6(The accession NO in GenBank is HQ156228). The gene encoded a Proteinof394amino acids with a deduced molecular weight about44.8kDa and PI of5.13.Analysis by real-time PCR indicated that the expression of BnMPK6was increased inresponse to cold stress.2. In this study, BnMPK6gene promoter which from Brassica napus (rape) longyou6was isolated by chromosomal walking.1337bp sequence was obtained by sequencing (GenBank accession number for the JN830898). The functional elements wereanalyzed by Plant CARE. Bio-informatical analysis of this sequence showed that thepromoter contains some typical elements: CAAT-box, and stress-induced elements:chill-proof and ABA stress responded element, chill-proof, ABA and drought controlresponded element, cis-acting anaerobic regulatory element, gibberellin-responsiveelement and cis-acting low-temperature responded element. Furthermore, there was aMYB binding site involved in drought-inducibility MBS in the promoter. Sequenceanalysis indicated that the promoter was a strong adversity stress-induced promoter,especially in cold and drought.3.A experiment of Brassica napu seedings (Tianyou2and Longyou6) under lowtemperature stresss showed that the chlorophyll contents and the maximum rate ofphotochemistry (Fv/Fm) reduced, the contents of MDA increased, while the activitiesof CAT, APX, GR and SOD increased first and then decreased. MAPKK inhibitorsU0126pretreatment decreased chlorophyll contents and Fv/Fm, as well as variousantioxidant enzyme activities, while MDA contents increased. In conclusion, MAPKcascades take part in the transduction pathways of low temperature stress and play apositive role in antioxidative damage.