Identification And Functional Analysis Of Key Genes Regulating Prickle Development Of Leaves In Zanthoxylum Armatum

Zanthoxylum armatum var.novembolius(Z.armatum),a cultivated variety of Zanthoxylum armatum DC.,belongs to Zanthoxylum of Rutaceae.Z.armatum has become an important economic tree species because of its unique taste characteristics and potential medical use.The prickles of Z.armatum are all over the tree,which seriously restricts the development of Z.armatum industry.The prickles of Z.armatum have always been a relatively thorny problem for pepper farmers in the process of planting and cultivation management of Z.armatum.Researchers hope to fundamentally solve this problem by cultivating few prickly or pricklless varieties of Z.armatum.At present,breeders mainly obtain few prickles or pricklless varieties through the introduction of excellent varieties and asexual reproduction such as grafting to obtain few prickles or pricklless varieties.However,the grafting propagation method is not easy to be widely popularized.And there is a serious atavism phenomenon in seed propagation,that is,the offspring will show the phenotype of their ancestors with prickles,which is impossible to obtain few prickles or pricklless varieties with stable inheritance.Moreover,there are few reports on molecular mechanism of prickly development of Z.armatum.Therefore,the high-quality full-length transcript database of leaves in different development stages of Z.armatum(prickly)and its budding variety Z.armatum ‘Rongchangwuci’(pricklless)was constructed through third-and second-generation sequencing technology,which provided high-quality gene resources for the research of Z.armatum phenotype.Based on this,the key genes in the development of prickles were excavated,and the potential molecular mechanism of Z.armatum prickles formation was analyzed,which laid a theoretical foundation for using the genetic engineering to breed Z.armatum varieties with few prickles or pricklless varieties.The main results of this study were as follows:(1)In this study,leaves of Z.armatum(ZY)and its budding variety Z.armatum‘Rongchangwuci’(WC)in three different development stages(10,60 and 120 days after germination)were used as research materials.The Pac Bio platform was used to sequence the full-length transcriptome,16.26 Gb clean data,533,462 reads of insert(ROI)sequence and 392,116 full-length non-chimeric sequences(FLNC)were obtained in the WC;and 15.22 Gb clean data,456,134 ROI sequences and 355,210 FLNC sequences were obtained in the ZY.Further cluster analysis of the full-length(FL)sequence,208,933 and 187,358 consistent transcript sequences were obtained from ZY and WC,respectively.The transcriptome databases of ZY and WC at three different development stages were constructed,and 117.65 Gb of Clean reads were obtained.The non-full-length sequences were used to correct it,and 137,429 and130,588 high-quality transcript sequences were obtained,respectively.The secondgeneration data was used to correct the third-generation low-quality data.And 148,445 transcript sequences were obtained.From which,105,465 simple sequence repeat(SSR),114,205 complete coding region sequences and 4,719 lnc RNAs were prcdicted.And 6,647 and 3,795 alternative splicing events(AS)were predicted from ZY and WC,respectively.A total of 96,931 differentially expressed genes(DEGs)were identified among different comparison groups,of which 94,341 DEGs were annotated.The contents of auxin,cytokinin,jasmonic acid and abscisic acid in the samples were determined.To understand the role of plant hormones in the formation of prickles,the contents of jasmonic acid,auxin and cytokinin in the leaves of ZY were significantly higher than those in the leaves of WC,and there was no significant difference in abscisic acid.According to the high-quality transcript data and gene annotation information,1,560 DEGs might be involved in regulating the metabolic processes of auxin,cytokinin,jasmonic acid and abscisic acid.Combined with weighted gene coexpression network analysis,eight genes(MYC,IAA,ARF,CRE/AHK,PP2 C,ARR-A,AOS and LOX)were identified,including 25 transcripts,which might affect the metabolism of auxin,cytokinin and jasmonic acid,and indirectly participate in the formation of prickles of ZY.Among them,ARF5.2 in auxin metabolic pathway had an AS event,and 20 genes out of 25 genes were regulated by 69 lnc RNAs,which indicated that the AS event and lnc RNA might be involved in regulating the development of prickles.The results of this study also showed that the combined analysis of third-and second-generation sequencing of full-length transcriptome could play an important role in grasping the genetic information of species lacking reference genome information or incomplete genome information,and clarifying the genetic regulation mechanism related to plant life activities.(2)Many transcription factors(TFs)are involved in the development of plant trichome.In order to identify the key transcription factors involved in prickle development in Z.armatum,a total of 217 differentially expressed TFs were screened out,including 170 MYBs,9 b HLHs and 38 HD-ZIPs.Combined with the genes successfully reported in other plants to regulate the development of epidermis,eight key TFs related to prickle formation of ZY were identified,including six MYBs,one GL3 and one GL2.Bioinformatics analysis showed that the eight proteins were all unstable and hydrophilic proteins without transmembrane domain,and all of them had phosphorylation sites.The results of conserved domain analysis showed that MYB1,MYB2,MYB3,MYB4 and MYB5 belonged to R2R3 MYB TFs,GL3 belonged to HDZIP TF family,and GL2 belonged to b HLH TF family.And the expression patterns of eight genes in different tissues were analyzed,and all of them were tissue-specific.(3)The research on the function of key candidate TFs for prickle formation in Z.armatum showed that the heterogenous expression of Za MYB86 in mutant A.thaliana significantly reduced the number of trichomes,which indicated that Za MYB86 inhibited the development of trichomes in A.thaliana,and it was speculated that Za MYB86 might be the negative regulatory factor of prickle development.In order to analyze the potential molecular mechanism of Za MYB86 regulating the development of prickles of ZY,this study predicted the interaction between Za MYB86 and Za MYB5,and the interaction between Za MYB86 and Za MYB5 was verified via yeast two-hybrid technique and bimolecular fluorescence complementary experiment.Comparison shows that the MYB DNA binding domain of Za MYB5 was highly similar to that of At MYB5 in A.thaliana,and At MYB5 is the negative regulatory factor of trichome development.Therefore,this study puts forward the potential molecular mechanism that Za MYB86 and Za MYB5 interact and jointly participate in the regulation of prickle formation of Z.armatum.