Study On The Involvement Of Banana ACC Oxidase Gene During Fruit Ripening

Banana(Musa acuminata)belongs to the monocotyledonous plant of the genus Musa of the Musaceae family,and is a typical climacteric fruit.As one of the important agricultural crops in tropical and subtropical regions,bananas are often overripe and spoiled due to the rapid increase in respiration rate and ethylene production during transportation,which restricts their transportation,storage and sales,and affects the market value and popularity of bananas.Ethylene biosynthesis is affected by the efficiency of ACC oxidase conversion to ACC.In previous studies,MaACO1 and MaACO2 genes were screened out of 18 ACC oxidase homologous genes in the banana genome,which were mainly involved in fruit ripening.Using Taiwan banana cultivar ’Pei-Chiao’(AAA type,Cavendish subgroup)as the material,the construction of MaACO1 and MaACO2 as the target gene expression vector of si RNA hairpin structure,and the transgenic banana plants were obtained by Agrobacterium-mediated method.By inhibiting the biosynthesis of ethylene,the purpose of delaying fruit ripening is achieved.In order to deeply understand the complexity and diversity of functions between MaACO1 and MaACO2,this paper integrated and analyzed the differential expression profiles of miRNA sequencing and mRNA transcriptome data of Beijiao wild-type(WT),MaACO1 and MaACO2 transgenic bananas(As1 and As2)at the third maturation stage.In this paper,we screened and identified the expression patterns of mRNA,miRNA and their target genes involved in the inhibition of climacteric changes during fruit ripening due to the inhibition of ethylene synthesis pathway in banana through fluorescence quantification,hormone treatment,race cloning,yeast single and double hybrid and other experimental means.The main research results and conclusions obtained in this paper are as follows:1.Transcriptome analysis of WT,MaACO1 and MaACO2 silenced transgenic banana fruitCompared with wild-type banana fruit,RNAi transgenic banana fruit significantly inhibited ethylene release during post-ripening process,and As2 was more obvious than As1 in banana fruit ethylene inhibition.Combined with transcriptome data obtained from transgenic and wild-type banana fruits at the third maturation stage,it was shown that inhibition of ethylene production would lead to down-regulation of sucrose metabolism pathway related genes such as sucrose synthase(SUS),sucrose phosphate synthase(SPS)and neutral sucrose invertase(Inv-N1),and As2 was more obvious than As1 in inhibiting sucrose hydrolysis.At the same time,the inhibition of ethylene biosynthesis led to the obvious down-regulation of the expression of genes related to the carotenoid synthesis pathway,such as phytoene synthase(PSY)and Zeta-Carotene desaturase(ZDS),and the carotenoid accumulation in the pulp of As2 relative to As1 decreased significantly.It is proved that MaACO2 plays a leading role in regulating the metabolic pathways related to ethylene biosynthesis and fruit maturation.2.Screening of MaACO2 binding protein and its promoter-interacting transcription factors in banana fruitIn this study,a yeast library was constructed for banana fruit pulp at the third stage of ripening.Through yeast two-hybrid experiments,27 proteins that interacted with MaACO2 were preliminarily verified.Among them,chitinase 1(Chitinase 1)and endo-1,3-β-glucosidase(endo-1,3-β-glucanase)were further identified to participate in the interaction of MaACO2 protein.And it was found that the blockage of ethylene synthesis will cause the expression of chitinase 1 and endo-1,3-β-glucosidase to be significantly inhibited.It was speculated that chitinase 1 and endo-1,3-β-glucosidase,combined with MaACO2 protein,may positively regulate banana fruit maturation.Secondly,in this study,the full length of the MaACO2 promoter was used as the bait plasmid,and the proteins bound to it were screened out from the library.40 annotated proteins were obtained through rescreening and sequencing,and it was further confirmed that NAC 21/22 and RING-H2 zinc finger protein ATL2 participated in the interaction of the MaACO2 promoter respectively.At the same time,it was found that the expression of NAC21/22 gene was significantly down-regulated and the expression of ATL2 gene was significantly up-regulated when ethylene synthesis was significantly inhibited.It is speculated that NAC21/22 and ATL2 transcription factors may positively and negatively regulate banana fruit ripening,respectively.3.Differential analysis of miRNA in WT,MaACO1 and MaACO2 silenced transgenic banana fruitAccording to the small RNA sequencing data of RNAi silencing transgenic banana fruit stage 3,the clean reads for subsequent analysis were 6912621(90.20%),8337765(94.06%)and 7457309(88.76%)in WT,As1 and As2,respectively.Among the 44 significantly different genes,miR390b-5p and miR172 a were significantly up-regulated when the expression of MaACO1 and MaACO2 was inhibited,suggesting that both of them were involved in ethylene biosynthesis.While miR156e-5p and miR319c-3p were significantly up-regulated only in As2,and miR164a-5p was significantly up-regulated only in As1.It was speculated that the differential expression of these miRNAs might be the key to differential regulation of ethylene synthesis and fruit ripening by MaACO1 and MaACO2.The predicted miRNA target genes are mainly enriched in five KEGG pathways,including sulfur transport system,protein digestion and absorption,histidine metabolism,pathogenic E.coli infection,sulfur metabolism,and starch and sucrose metabolism.It was also found that the ethylene synthesis pathway and ethylene signal transduction pathway were not enriched with related miRNAs and target genes,but the expression of genes related to the sulfur metabolism pathway upstream of the ethylene synthesis pathway was significantly up-regulated in the As1 and As2 samples.However,in the two samples,it was found that the expression of miR172 a was up-regulated and inhibited the expression of sulfur reductase and serine acetyltransferase,respectively,It is speculated that there is a feedback regulation between the sulfur metabolism pathway and ethylene biosynthesis.4.Expression pattern of miRNAs associated with ACC oxidase silencing and preliminary construction of banana regeneration systemIn order to further analyze the differential expression of miRNAs(miR169 a,miR319c-3p,miR171 a,miR156e-5p and miR164a-5p)and their target genes caused by ethylene biosynthesis inhibition during fruit ripening,banana fruits were treated with ethylene and 1-MCP respectively.It was found that miR169 a,miR319c-3p,miR171 a,miR156e-5p and miR164a-5p decreased with the ripening of banana fruits,and the down-regulation degree under ethylene treatment was significantly faster than that under 1-MCP treatment,it is speculated that these miRNAs may negatively regulate fruit ripening by regulating their corresponding target genes.At the same time,cluster analysis revealed that some powerful gene families expressed a large number of genes in banana tissues,such as MIR168,MIR166,MIR156,MIR159,MIR172 and MIR162.It was speculated that the highly abundant miRNAs expressed in bananas generally ran through the whole banana biological development and aging process.In addition,in order to further understand the expression of Pri-miRNAs and mature miRNAs during fruit ripening,this study used RACE technology to clone the full length of banana Primary of miR169 a and miR319c-3p,confirmed their transcription start sites,predicted their potential ORFs,and found that the expression of Pri-miR169 a and Pri-miR319c-3p increased gradually with the fruit ripening process,which was opposite expressed with their mature miRNAs during fruit ripening.At last,this study made a preliminary attempt at banana regeneration system by using immature male flower buds,screened the best adventitious bud induction medium,and confirmed the location of male flower nodes for optimal adventitious bud induction.By constructing gene editing vectors pGSfi1-sg156-gfpgus and pGSfi1-sg164-gfpgus targeting miRNAs related to fruit maturation,we hope to obtain transgenic banana plants by bombarding adventitious buds with gene guns.