Identification Of Citrus PG Genes And Their Roles In Fruitlet Abscission

Citrus is one of the important fruit trees and widely grown in China due to high economic value and rich nutrition.In recent years,citrus has become an important economic source for farmers to get rid of poverty in some areas,and play an important role in Rural Revitalization.Citrus industry having got remarkable achievement in China,but the excessive flower and fruitlet abscission has negatively affected yield and reduced the income of farmers.Therefore,it's very important to illustrate the molecular mechanism of Citrus flower and fruit abscission,with an aim to provide effective measures to resolve this problem.The process of plant organ abscission mainly includes cell separation and programmed cell death.PGs?polygalacturonases?belonging to pectin hydrolases,can modify and hydrolyse cell wall,and then result in cell separation,showing an important role in abscission.However,PG gene family has not been reported in citrus.Based on the clementine and sweet orange genomes,it is the first time for PG gene family in citrus to been analyzed in this study including physico-chemical characterstcs,gene structure,conservative domain,chromosome location,phylogenetic relationship,and the expression patterns in different tissues and during citrus fruitlet abscission.The CitPG34gene and its promoter?CitPG34-P?sequences were cloned from‘Tarcocco'blood orange?Citrus sinensis L.cv.‘Tarocco'?.By means of gene expression,genetic transformation and subcellular localization,the biological function of Cit PG34 in citrus fruitlet abscission?CFA?was preliminarily analyzed.The main results are as follows:1.Based on citrus genomic sequences and using Arabidopsis PGs as the reference sequences,38 members of citrus PG gene family were identified for the first time,which were named CitPG1-38,respectively.The result showed that the physico-chemical characterstcs among different CitPGs were similar,the ORF length varied from 378 to 2418bp encoding proteins with theoretical pI and the molecular mass ranging from 4.83 to 9.92and from 13951.71 to 85542.28 D,respectively.Most Cit PGs contained no less than 3 introns,suggesting a high probability of alternative splicing.The conserved domain analysis revealed that most of the members of CitPGs contained four typical conservative domains SPNTDG,GDDC,CGPGHG and RIK.Phylogenetic tree revealed that all PGs could be divided into three groups,which indicated that most CitPGs gene highly conserved during evolution,but there were differences in the functions of the members.Among the 38 CitPGs,in which 9CitPGs,including Cit PG2,CitPG3,CitPG10,CitPG24,CitPG27,CitPG29,CitPG30,CitPG33 and CitPG34 possessed a close relationship with abscission-associated PGs,indicating their role in CFA.RT-qPCR analysis further demonstrated that CitPG2,CitPG29and CitPG34 might be involved in CFA,the expression levels of which could be induced by ethylene,inhibited by IAA and increased during CFA.2.According to the abovementioned data,CitPG34 was selected for further analysis.We cloned it from‘Tarcocco'blood orange and found its ORF consisted of 1194 bp,encoding397 amino acids.The molecular weight of Cit PG34 was 41472.60 D,the theoretical p I 5.19,and the instability coefficient 30.23,indicating that CitPG34 belonged to stable protein.TMHMM analysis showed that CitPG34 was a transmembrane protein,the transmembrane domain locating between the amino acid residue 7 and 29.In the secondary structure of CitPG34,the alpha-helix structure,extended chain and random coil account for 15.37%,29.72%and 54.91%,respectively,which were nearly consistent with its tertiary structure.CitPG34 contained four typical PG conserved domains i.e.SPNTDG,GDDC,CGPGHG and RIK.NJ phylogenetic tree showed that CitPG34 with abscission-related PGs,for example tomato TAPG1,TAPG2 and TAPG4,was clusted into A branch,closest to pear PcPG3?BAF42034?,indicating that it might be related to fruit abscission.Tissue-specific expression analysis showed that CitPG34 dominantly expressed in flowers,followed by roots,leaves,abscission zone A?AZ A?and C?AZ C?,and almost undetected in fruits.Subcellular localization revealed that CitPG34 was mainly located in cell wall.Overexpressing CitPG34in tobacco to obtain T₀ transgenic plants,which provided a test material for further research.3.A 2075 bp promoter sequence of CitPG34 was cloned,which contained several cis-regulatory elements responded to light,drought,anaerobic stress,fungal induction and hormone?abscisic acid,jasmonate and gibberellin?,etc.CitPG34-P::gus expression vector was constructed and transformed into tobacco.GUS histochemical staining revealed that the GUS activity in vein was remarkably up-regulated by ethylene.