Cloning And Preliminary Functional Analysis Of SWEET Gene Family In Litchi Chinensis

Litchi(Litchi chinensis Sonn.)is an evergreen fruit crop.The aril contains a lot of soluble sugar in litchi.Based on different ratios of hexose/sucrose,sugar composition in the aril of litchi fruit varies greatly among cultivars.Different litchi cultivars can be divided into disaccharide-prevalent cultivar(’Wuheli’),intermediate type(‘sanyuehong’),monosaccharide-prevalent cultivar(’Feizixiao’).Moreover,the ratio of hexose/sucrose was significantly positively correlated with the embryo abortion in litchi.According to the abortion of embryo,litchi can be divided into three types: big-seeded cultivars(‘heiye’),aborted-seeded cultivars(‘nuomici’),and partly aborted-seeded cultivars(‘lanzhu’).The nectary of litchi belongs to starch type,and the main components of nectar are sucrose,glucose and fructose.SWEETs(Sugar Will Eventually be Exported Transporters)function as sugar efflux transporters that have diverse physiological functions,including nectar secretion,sugar accumulation,and seed development.The SWEET gene family has been identified in many plant species,but little is known about in Litchi.In this study,we cloned and comprehensively analyzed the SWEET gene family in litchi.In addition,we identified the LcSWEET gene involving in aril sugar accumulation,seed development,and nectar secretion by gene expression analysis.Our research lays a foundation for the elaboration of the functions of the LcSWEET genes in litchi.The main results are as follows:(1)Sixteen LcSWEET genes with two Mt N3/saliva domains(Pfam motif pf03083)were screened and cloned from Litchi genome.The LcSWEET genes family were divided into four clades(Clade I to Clade IV)by phylogenetic tree analysis.Moreover,LcSWEET genes in the same Clade share similar gene structures in terms of exons location and introns length.Multiple sequence alignment of the 16 LcSWEET full-length protein sequences showed that the 7-TMs and motifs were basically conserved in specific positions.There was one pairs of segmental duplicated events and 3 tandem duplication events in the LcSWEET gene family.(2)In different tissues(leaves,stems,roots,male flowers,female flowers,pericarps,arils,and seeds),the expression patterns of the LcSWEET genes showed higher expression levels in reproductive organs by qRT-PCR analysis.(3)qRT-PCR was used to analyze the expression of LcSWEET genes during litchi aril development between ‘Feizixiao’ and ‘Wuheli’.The results showed that the expression pattern of LcSWEET10 gene was correlated with the sugar accumulation pattern in litchi.Additionally,the expression of LcSWEET10 in ‘Feizixiao’ which accumulated more hexose was lower than that in ‘Wuheli’ which accumulated more sucrose.It was speculated that LcSWEET10 might be involved in the sugar accumulation in litchi.(4)qRT-PCR was used to analyze the expression patterns of LcSWEET gene in the early stage of seed development between big-seeded ‘Heiye’ and aborted-seeded ‘Nuomici’.It was found that a peak expression of LcSWEET2 a at ‘Heiye’ was earlier than ‘Nuomici’.The expression of LcSWEET3 b increased significantly after ‘Heiye’ 20 DAA,but only slightly in ‘Nuomici’.The results of RNA in situ hybridization showed that LcSWEET2 a and LcSWEET3 b were highly expressed in the funicle and ovule of ‘Heiye’ and very low in‘Nuomici’,which indicated that LcSWEET2 a and LcSWEET3 b played an important role in the early stage of litchi seed development.(5)qRT-PCR analysis of LcSWEET gene expression in 9 flower tissues showed that LcSWEET9 a and LcSWEET9 b were highly expressed in nectary of male and female flowers.Subcellular localization analysis showed that both LcSWEET9 a and LcSWEET9 b were located in the plasma membrane.The transformation of LcSWEET9 a and LcSWEET9 b genes mediated by Agrobacterium tumefaciens could not complement the function of Arabidopsis mutant(Atsweet9)in nectar secretion.However,the silencing of LcSWEET9 a and LcSWEET9 b genes in ‘Feizixiao’ significantly reduced the nectar secretion,indicating that LcSWEET9 a and LcSWEET9 b played an important role in nectar secretion in litchi.