Cloning And Functional Validation Of Five Abiotic Stress Inducible Promoters In Maize

The promoter is the DNA sequence of initiating gene transcription, which located in the front of gene sequence and is a key point for regulation of the transcriptional level. Inducible promoter has lower transcriptional activity or doesn’t initiate gene transcription without the effect of inducible factor, but it would initiate gene transcription and increase transcriptional activity dramatically with the effect of inducible factor.The tolerance of maize to abiotic stress shows a complex heredity, and it is difficult to identify it. For many years, there is little breeding effectiveness. Importing exogenous resistance gene through transgenic technology can significantly improve maize resistivity to abiotic stress. However, some constitutive stronger promoters-ubiquitin, CaMV35S, actin-could initiate the overexpression of resistance gene in plant resistance gene transgenic research. Although the resistivity of the transgentic plant can be improved, the overexpressions of resistance gene could inhibit plants physiological and metabolic activity, and even seriously affect the normal growth and development of the plant, resulting in slow growth, dwarf plant and low yield for transgenic plants compared with wide type plants. Inducible promoter could initiate expression of resistance gene in adversity induced conditions or the particular stage of plant growth, so that the plants could resist adversity stress but also could grow normally in non-stress conditions, and reduce the price of the adaptability generated by overexpression of exogenous resistance gene.In this study, genes of HVA1, HVA22, ABA3, DREB1B and ABP9initiated by inducible promoters which had been validated in Arabidopsis, barley and rice were used as probes, and blasted them with the sequence of the maize inbred line’B73’to find out their homologous genes MGL3, HVA22, ABA3, sbCBF6and ABP9in maize, and also concluded and cloned1500bp as their promoters from their upstream sequences, and predicting the cis-acting regulatory element of promoter by PlantCARE software. Replace the CaMV35S promoter of PBI221vector with the cloned sequence, and construct the transient expression vector. The constructed vectors were transformed into maize callus using biolistic (gene gun) method and detecting the activities of cloned promoters by histochemical staining method. It could produce4-MU when take4-MUG as the substrate and reacted with GUS enzyme under high-salinity, high permeability, ABA, cold treatment and detecting the fluorescence of4-MU by porous detector. Then calculated the expression level of GUS gene and compared them with the expression level of GUS gene without treatment. Extracted mRNA of maize seedling subjected to drought, high-salinity, ABA, ethylene, cold, heat treatment at different time periods and analyzed the expression difference of MGL3, HVA22, ABA3, sbCBF6, ABP9genes under induction conditions by qRT-PCR, then compared this result with that of fluorescence detection.MGL3s contains multiple ABRE elements involved in ABA-inducibility and some cis-acting regulatory elements involved in osmotic pressure regulation such as ARE, CCAAT-box, MBS, CGTCA-motif, TGACG-motif and so on. MGL3s initiated GUS expression and transformed them into maize callus, then detected GUS expression level with histochemical staining method. Taked X-GLUC as the substrate, MGL3s had initiating activity and could initiate GUS expression, the callus where GUS gene expression in would produce blue dot after staining. The activity of MGL3s could be detected by fluorescence. Its expression level was very low without treatment and significantly improved under high permeability, showing obvious difference. qRT-PCR analysis showed that MGL3s was also can be induced under drought, high-salinity, ABA, ethylene, cold, heat treatment.sbCFB6s contains multiple cis-acting regulatory elements involved in ABA, drought, trmperature regulation such as ABRE, ARE, AT-rich sequence, Box-W1, CGTCA-motif, HSE, MBS and so on. sbCFB6s showed obvious difference induced by cold stress, its expression level was increased under high permeability, high-salinity and ABA treatments, but the rate of increase was less than that under cold stress. qRT-PCR analysis showed that sbCFB6s expression was increased under cold and heat stress, this result was indentical to that detected by fluorescence.ABA3s contains cis-acting regulatory elements of ARE, HSE and MBS. ARE was involved in osmotic pressure regulation, HSE was involved in temperature regulation and MBS was involved in drought stress. ABA3s showed the most obvious difference in expression under high permeability detected by fluorescence and its expression was decreased greatly under cold stress. qRT-PCR analysis showed that ABA3s expression was increased under ethylene and high-salinity treatments and reduced under cold stress. This was basically the same as the result of fluorescence detection.HVA22s has higher expression level normally, but its expression was inhibited by high-salinity, high permeability, ABA and cold treatments, this was indentical to the result analyzed by qRT-PCR. ABP9s expression was increased under various stresses detected by qRT-PCR. After verification, MGL3s could be used for genetically modified maize experiment and initiated the expression of drought-resisting gene and salt tolerant gene, ABA3s could be used to initiate the expression of salt tolerant gene, sbCFB6s could initiate the expression of low temperature resistant gene. They could make genetically modified organism maize resist adversity stress and let them grow and develop in normal conditions without adverse effects.