Functional Dissection Analysis Of The Promoter Of Myrosinase Associated Protein Gene

In the Cruciferae plant there is a special substrate - enzyme system, namely glucosinolate -myrosinase system. In vivo, Myrosinases usually occur in complexes with myrosinase-bingingprotein and myrosinase-associated protein, iMyAP is one of myrosinase-associated proteinspresent in vegetative organ, which can be promoted by iMyAP promoter induced by wounding,MeJA, JA and the ABA treatment.In order to further understand the function- structure characteristic of iMyAP genepromoter, to identify the size of core promoter of iMyAP promoter, we divided the whole-1868bp promoter into 5 fragments, inserted the 5 fragments and whole promoter just beforeGUS gene and transferred the fusions into Arabidopsis genome. Then the functional structure ofiMyAP gene promoter was analyzed on the arabidopsis heredity background.The results showed that the methyl jasmonnic acid(MeJA) and wounding processing canmake iMyAP promoter propel the GUS gene expression in the whole plant. The up-ground wasstained deeper than the underground and the vascular tissue deeper than the parenchyma. Thejoint area of leafstalk and root was stained the deepest.370bp fragment is enough for finishing the induced function of whole promoter by deletionanalysis. M4 - 5 with 370bp fragment. can start up the GUS gene expression after being treatedby wounding and methyl jasmonic acid and the blue position and blue intensity is consistent withone of M4-1. When the whole promoter was deleted to 153bp, the wounding and the jasmonicacid treatment cannot induce the GUS gene expression in M4-6, which means that this 153bpDNA sequence does not have the induction function after treated by wounding and MeJA. Byuse of the the PLACE, we analyzed the motif in the 370 fragment and found that the fragmentaccounting for 19% of whole promoter has all kinds of 8 motifs relative to induction of wholeiMyAP promoter and includes a sole JA-responsive motif in the whole promoter; a soleauxin-responsive motif, a sole sugar-responsive motif, one of two wounding-responsive motifs,three of four anaerobically-responsing motif, all 6 type motifs relative to dehydration-responsive(among the 6 type motifs MYCATERD1 and MYCATRD22 are sole). These data and the resultsof GUS histochemical stain together fully showed that the 370bp fragment has represented thebasic characteristic of induction function of iMyAP promoter, namely the cor promoter of theiMyAP promoter. This is the first time to identified the size of the iMyAP core promoter.Not only the 370bp core promoter can start the gene expression after wounding and MeJAtreatment, but also the core promoter has the characteristic of cell-specific promoter. The corepromoter can start the GUS gene expression specifically in the guard-cell under normal growthcondition, ands act as a typical tissue-specific promoter. But all known tissue-specific motifs arenot found in the 370bp core promoter. Under the ostensible contradictory phenomenon, it is hintsthat there is an unknown guard-cell-specific motif in the core promoter. Further experimentalneed to be done for this deduction. This phenomenon has not been reported until now that the new promoter from the primary promoter deletion not only maintains the function of primaryiMyAp, but brings novel promoter function.The GUS quantitative analysis results from M4-1 to M4-6 demonstrated that at less there isan enhance element between - 1868～- 1506bp(363bp), because the 363bp loss caused GUSactivity decrease in larger scale. It is first time to find enhance element in iMyAP promoter.The core promoter can propel the GUS gene expression in guard cell under highertemperature stress and normal growth condition. But based on the stained guard cell numbers,the blue guard cell numbers are fewer under higher temperature than normal temperature. It issuggested that higher temperature inhibits the GUS gene expression promoted by 370bp in guardcell.The core promoter can also propel the GUS gene expression in guard cell under lowertemperature stress and normal growth condition. But the stained guard-cell numbers are fewerunder lower temperature stress than normal growth condition. No known motif related to lowertemperature response had been found in the 370bp by PLACE. So further deletion analysis of thecore promoter will be helpful to find new motif responsive to lower temperature stress.At the root tip of M4-1 treated by lower temperature treatment, deeper GUS dye werealong the vascular bundle and in two central cell of the third story columella cell(S3), and theparenchyma tissue around the vascular bundle and the elongation zone were stained nothing.Opposite the curving direction, in the elongation zone, half central cylinder was stained blue,another half wasn't stained. Speaking of the S3 columella, two have the blue color, but the cellopposite curving direction was obviously stained more intensive and the size was biggercompared to another one. According to the two observation facts, it is inferred that iMyAPpossibly participates in the response of root gravitropism. This extrapolation waits for the furtherexperimental confirmation.