Passion Fruit NAC Gene Resistance Response To Pathogenic Bacteria And PeNAC063 Function Verification

Passion fruit(Passiflora edulis Sims.)is rich in nutrients,not only sweet and sour,but also has a variety of biological functions.Because it has the aroma of a variety of fruits,and is rich in flavonoids,alkaloids and other active compounds,it is known as the "king of fruits".However,due to its postharvest vulnerability to microbial infection,the storage tolerance of the fruit is reduced,which seriously affects the product value.Moreover,the laboratory research group has successfully isolated and identified highly infectious pathogenic fungi such as F.kyushuense and A.alternate in passion fruit,so in order to improve the quality of passion fruit,it is urgent to study the process and mechanism of pathogenic bacteria infection with passion fruit.As one of the largest families of transcription factors in plants,NAC plays an important role in participating in plant resistance to stress stress and is particularly significant in regulating biological stress.In order to reveal the mechanism of NAC family members in regulating the infection of passion fruit by pathogenic bacteria,this study used ’Fujian passion fruit No.1’purple fruit and ’Fujian passion fruit No.3’ yellow fruit as experimental materials,and used F.kyushuense and A.Alternate to infect two varieties of passion fruit.The contents of ABA,SA,JA and MeJA of passion fruit peel and the change of antioxidant capacity of passion fruit peel after bacterial infection were determined by high performance liquid chromatography and DPPH method.Bioinformatics analysis was performed on members of the passion fruit NAC family and qRT-PCR was used to analyze the relative expression of related PeNACs under passion fruit biostress.The pCAMBIA1301-PeNAC063 overexpression vector was constructed,and the functions of PeNAC063 were explored and verified by transient transformation of passion fruit and genetic transformation of passion fruit seedlings by injection method and in situ technology transformation.The main findings are as follows:1.Identification of PeNAC transcription factors and their expression under passion fruit pathogen stressIn the passion fruit genome,105 PeNAC genes were screened and identified,which encoding amino acid lengths ranging from 120-3052 bp,including 3-8 conserved motifs and 1-3 introns.Subcellular localization predicts that most of the PeNAC protein is localized in the nucleus.It is named according to the location of chromosomes,PeNAC001-PeNAC 105 and classified according to the phylogenetic tree,which can be divided into 15 subgroups of NAC-a～NAC-o.All PeNACs contained homeopathic elements for plant growth and development,light,hormones,and stress responses,and all PeNAC proteins exhibited similar 3D structures.During the evolution of PeNAC genes,a total of 11 tandem repeat pairs and 12 fragment repeat gene pairs were identified.In addition,17 predicted ped-miRNAs were identified as targeting 25 PeNAC genes,and potential transcription factors including ERF,BBR-BPC,etc.were identified in the promoter region and interactions with PeNAC genes were predicted.Preliminary screening of 15 PeNAC genes for qRT-PCR assay analysis showed that PeNAC001,PeNAC003,PeNAC008,PeNAC028,PeNAC033,PeNAC058,PeNAC063 and PeNAC077 genes were significantly upregulated compared with the control under Fusarium infection.The eight PeANCs excavated above laid the foundation for the study of the response function of passion fruit to biological stress such as Fusarium Kyushuense and Alternaria alternata.2.Response of PeNAC-related transcription factors to endogenous hormone and antioxidant levels under passion fruit pathogen stressYellow fruit and purple fruit produce obvious changes in fruit appearance under fungal infestation.Compared with yellow fruit,the plaque growth diameter of purple fruit is significantly smaller,and the degree of decay is also less than that of yellow fruit.However,in general,a little hyphae growth was seen in 3-6 dpi,while a large amount of hyphae grew at the inoculated fungus site after 9-12 dpi and even covered the entire fruit surface;And the peel is wrinkled,the fruit weight decreases significantly,and the fruit rots.In addition,with the increase of infection time,the TSS content of passion fruit decreased significantly,while the pH value of passion fruit increased slightly.The results showed that except for the trend of MeJA,MeJA first falling and then rising and then declining,the other three hormones showed that the other three hormones showed an upward and then downward trend after fungal infection,but the content of purple fruit was lower than that of yellow fruit.The SA,JA and ABA contents of yellow fruit reached a maximum of 8.61 ng/ml,10 ng/ml and 55.48 ng/ml in 9 dpi,while that of purple fruit reached a maximum of 7.46 ng/ml and 38.10 ng/ml at 12 dpi,while jasmonic acid reached a maximum of 2.69 ng/ml in 9 dpi.Compared with the control,the antioxidant capacity of yellow fruit and purple fruit increased first and then decreased,and also reached a maximum at 9 dpi,but the difference was that the antioxidant capacity of purple fruit at 12 dpi was significantly greater than that of yellow fruit.In addition,the correlation analysis between the relative expression of 15 PeNACs and hormone levels and antioxidant capacity showed that the relative expression of PeNAC063 and PeNAC077,PeNAC078 genes,PeNAC085 and PeNAC063,PeNAC077 and PeNAC078 genes were significantly positively correlated.PeNAC0063,PeNAC007 and PeNAC028 were positively correlated with ABA and JA contents.3.Cloning of the PeNAC063 transcription factorAmong the 15 PeNAC genes,5 PeNAC genes that were stable and highly expressed after passion fruit inoculation with pathogenic bacteria,PeNAC001,PeNAC028,PeNAC058,PeNAC077 and PeNAC063,were screened.The nearest kinship of PeNAC063 was subsequently studied by multiplex amino acid sequence comparison with six transcription factors ATAF1,ATAF2,SlNAC1,OsNAC1,OsNAC48 and VvNACl,which have been identified as participating in the regulation of mechanisms in resistance to biological stress.The multiplex amino acid sequence alignment results showed that they were highly conserved at the N-terminus,and the sequence similarity was 44.81%.The results of protein phosphorylation showed that although the number of phosphorylation sites was different,the phosphorylation sites were almost similar,and the PeNAC063 was similar to the 2nd and 3D structures of six transcription factor proteins such as ATAF1 and ATAF2.In addition,the cDNA sequence of passion fruit PeNAC063 gene was successfully cloned with a sequence length of 1065 bp and encoding 354 amino acids.4.PeNAC063 transcription factor improves passion fruit disease resistance to F.kyushuenseThe passion fruit pCAMBIA1301-PeNAC063 overexpression vector was successfully constructed,and the transient expression function of purple fruit and yellow fruit ripe fruit was verified by injection.The results showed that passion fruit containing overexpression pCAMBIA 1301-PeNAC063 carrier Agrobacterium infestation was significantly higher than that of wild-type passion fruit after 2 days.After 5 days of inoculation with Fusarium Kyushu,purple fruit and yellow fruit that overexpressed PeNAC063 gene did not grow or the diameter of plaque was significantly smaller than that of wild type.There were also differences between the two varieties of yellow fruit and purple fruit,and the growth of purple fruit plaque was better than that of yellow fruit.Agrobacterium infestation solution containing pCAMBIA1301-PeNAC063 overexpression vector was infected with passion fruit seedlings by in situ technology transformation,and the transgenic germination rate of passion fruit purple fruit and yellow fruit was 26.12%,the positive rate of DNA level identification was that the relative expression of PeNAC063 of positive plants was significantly higher than that of wild-type passion fruit seedlings.