Characterization Of PAL Gene Expression And Response To Defense Gene Infection Of Powdery Mildew In Cucurbita Pepo

Hull-less Cucurbita pepo is a rare variation in the natural world.Because its seeds are seedless and unseeded,its appearance resembles that of peeled husks,so it is also called shellless squash.However,over a long period of time,the cultivation of naked kernel American pumpkin breeding technology has been too traditional.There have been problems such as low seed yield,unstable shell-free rate,susceptibility to mixed and unsatisfactory comprehensive commodity characters,and large-scale spread of pumpkin powdery mildew and virus disease.It seriously affected the yield and quality of pumpkins in the Americas.In the previous study,the PAL gene family members were cloned.In this study,N.americana was used as the experimental material to clone the promoter of PAL gene family members from the American pumpkin,and bioinformatics analysis was performed.At the same time,the expression of PAL gene family members at different developmental stages of shelled American pumpkin and naked American pumpkin plants at different developmental stages and the specificity of PAL gene,CCR gene,and 4CL gene organization in stems and leaves of American pumpkin after inoculation with powdery mildew The research is conducted in the following aspects.The main findings are as follows:1.Three specific primers were designed for the c DNA sequence of the PAL gene of Pleurotus cucurbitae obtained in the previous period.The promoter sequence with a length of 1089 bp was cloned,and the obtained sequence was compared with the PAL DNA sequence of the naked kernel pumpkin squash.There was a 317 bp repeat between the two sequences,and the preliminary sequence was determined to be the promoter sequence upstream of the CPL-PAL gene.The obtained promoter sequence was analyzed using bioinformatics software.The results showed that the promoter contained the essential element TATA-box and the enhancer CAAT-box required for the eukaryotic promoter,and also contained other arid elements and methyl jasmonate regulation.Components,Hormone Response Elements,Hyaluronic Acid Elements,and Tissue-Specific Expressions.The predicted results indicate that the cloned promoter sequence has the general characteristics of a typical promoter and is involved in stress induction such as light,hormones,and drought.2.After the inoculation of powdery mildew with different resistant varieties of American pumpkin,the overall expression of the five defense genes PAL-1,PAL-2,PAL-3,CCR,and 4CL increased first and then decreased.The gene expression level of defense genes in disease-resistant cultivars increased more than that in susceptible cultivars,and the gene expression level of defense genes peaked on the 5th and 9th days after resistance to cultivars in the cultivar of American cucurbits.The 9d reached its maximum value.The increase in the expression level of resistance genes was higher than that of the defense genes in susceptible varieties.Compared with the five genes of shelled varieties,the expression level of PAL-2 was higher than that of the other four genes.It was speculated that PAL-2 gene may play a major role in the resistance to powdery mildew in shell America pumpkin.The comparison of defense genes in bare-branches is the highest expression of PAL-1,suggesting that PAL-1 plays a leading role in the resistance of this variety.3.On the hull of the American pumpkin with inbred line 03N-123-6(P3),naked inbred line 1N304-7(P13),shelled inbred line 04LAg-26-2(P5)and bare inbred line 04LAg-26-28(P15)Real-time fluorescence quantification of PAL gene family members in different tissue sites of these 4 species.The results showed that the expression levels of PAL-1,PAL-2 and PAL-3 in seed coat of P3 and P5 were higher than that of P13 and P15 in each period,indicating that the synthesis of seed coat of N.americana may be affected by PAL gene family members.The regulation.Crusted American pumpkin P3,P5 and N.americana P13,P15 showed different expression levels in other tissues.P3 and P13 showed the highest expression level of PAL-1 gene in melon husk and the lowest was melon husk;in P5 and P15 is the most abundant expression of PAL-1 gene,followed by the seed kernel,and the least is the melon sac;P3 PAL-2 gene expression is the highest for the melon,the lowest melon,and P13 is the lowest For the melon sac,the lowest expression level of PAL-2 gene in P5 is melon;P15 is melon sac.The expression level of rind was higher in P3 and P13 than in the other three tissues.The PAL-3 gene expression level in P3 was the lowest in seed kernels;P13 was the melon bursa;the most active expression in P5 and P15 was melon husk,P5 The weakest is the kernel,and the melon pod is the least expressed in P15.