Functional Analysis Of The Pi63 Promoter Of Rice Blast Resistance Gene

Rice blast is one of the most serious diseases in rice,causing a large reduction in rice production worldwide.Discovering new resistance genes and breeding new disease-resistant cultivars are the most environmentally friendly and effective methods for controlling rice blast.The research on the molecular mechanism of rice resistance is an important basis for genetic improvement of rice resistance breeding.Pi63,on rice chromosome 4,showed broad-spectrum resistance to Magnaporthe oryzae in field trials.Our previous studies showed that the resistance of Pi63 is positively correlated with its expression level.The promoter plays important roles in gene expression,therefore studying the expression characteristics of the resistance gene promoter is an important way to elucidate the molecular mechanism of resistance.In this study,the promoter of the rice blast resistance gene Pi63 was analyzed.The results are as follows:?1?Molecular identification and copy number identification of pPi63:GUS transgenic plants were carried out,and GUS staining was performed on different tissues of single-copy plants.The staining results showed that GUS was highly expressed in leaves and sheaths,and expressed in branches and glume,but not in stems,roots and mature seeds.The above results indicate that the Pi63 promoter has tissue specificity.?2?Molecular identification and copy number identification of T₁ and T₂generation Pi63 transgenic plants were carried out.Single-copy plants were selected for inoculation experiments of Magnaporthe oryzae,expression of Pi63 and defense-related gene before and after inoculation was evaluated by qPCR.The results showed that the expression of Pi63 was up-regulated after infection by Magnaporthe oryzae,and reached the highest value at 36 h after infection,and was rapidly down-regulated between 36 h and 48 h,and then up-regulated again.The above results indicate that the Pi63 promoter is an inducible promoter that responds rapidly to the infection of Magnaporthe oryzae.?3?GUS staining was performed using the pPi63:GUS transgenic single-copy plants after hormone treatment.The staining results showed that GUS expression was significantly up-regulated under SA treatment,and was significantly down-regulated after MeJA treatment.There was no significant change in GUS expression before and after treatment with 6-BA,ACC and NAA.The above results indicate that Pi63 is mainly involved in SA and JA-mediated disease resistance pathways.?4?GUS staining of leaves of T₁ generation pPi63-P0?P1?P2?P3:GUS transgenic single-copy plants showed that all leaves were stained,and the degree of staining of different constructions was different.Staining of P1 was the deepest staining,staining of P0 is lighter than P1,and the degree of staining of P2 and P3 was similar.The above results indicate that there were positive regulatory elements?related to jasmonic acid response element?between-1367 and-2148 bp and negative regulatory elements?related to auxin response element?between-2148 and-2896 bp in the Pi63 promoter,respectively,to balance the resistance and production.