Sex Differentiation Transcriptome And Differentially Expressed Genes Analysis In Pumpkin

Pumpkin,a typical flower with dioecious plant,belongs to Cucubita(Cucubita ssp.)annual vine.The hybrid showed strong heterosis,and was required cross-pollination every year.Soit is necessary to manually cast males in hybrid production process,which takes time and effort.It is of great significance to explore the sex determination mechanism of pumpkin and provides the basis for the pumpkin cultivation techniques.In this study,1 cm young shoots of the first-leave time and 10-leave timein subgynoecious ‘2013-12' and androecious ‘9-6' of C.maxima were used as materials,The Illumina HiSeq transcriptome was sequenced and statistical analysis and quality analysis were performed on the sequencing data.The important genes related to sex differentiation were screened and function analysis of these genes were developed.The main findings are as follows:1.63.56 Gb of Clean reads were sequenced in transcriptomes between subgynoecious and androecious of C.maxima,and 131960 unigenes with an average length of 688 bp were obtained by sequence assembly and splicing.During the four developmental periods,a total of 496 genes were up-regulated and 375 genes were down-regulated.The differentexpresion genes annotated into the GO database were classified into molecular function,biological process,and cellular component,the synthesis of plant hormones such as ethylene,auxin,gibberellin,abscisic acid and brassinosteroids,and hormone signal pathways were found involved into plant sex determination.7205 unigeneswere classified into 176 signal pathways using KEGG analyisis,and protein synthesis,protein metabolism and signal transduction were found involved in plant sex determination 38 genes involved in phytohormonal synthesis and signal transduction pathways were selected as the critical genes for sex determination.Up-regulated genes in the male flowering stage involved 10 genes involved in ethylene synthesis and signal transduction,and involved in abscisic acid synthesis and signal transduction pathways.There are 2 genes for protein kinases,2 genes related to cytokinins,12 genes that encode the auxin-response protein AUX/IAA in the auxin signaling pathway,and 2 genes involved in the gibberellin synthesis pathway.The genes up-regulated in the female flower stage were seven genes related to ethylene,abscisic acid,auxin and cytokinin,respectively.A portion of the genes were selected for qRT-PCR expression and the validation results were consistent with RNA-Seq.2.The expression patterns of five ACC oxidase genes,CmACO1,CmACO2,CmACO3,CmACO4 and CmACO5 in different organs during the flowering period of subgynoecious material ‘2013-12' were detected.These five genes are expressed in roots,stems,leaves,female flowers,and male flowers.The CmACO1 gene has the highest expression in female flowers.The CmACO1 gene was further tested at different flowering sites,as well as at different developmental stages of pistil and stamen,as well as at different hormone expression patterns.It was found that with the increase of CmACO1 gene accumulation in female flower development,it was speculated that CmACO1 gene was involved in the development of female flowers.The expression level of CmACO1 gene increased significantly after treatment with ethephon and auxin,indicating that CmACO1 gene was regulated by both ethylene and somatotropin.The full-length cDNA sequence of CmACO1 was 936 bp,and the bioinformatics analysis of CmACO1 was performed.3.The expression patterns of three genes CmAP2-1,CmAP2-2 and CmAP2-3,in different organs during the flowering stage of subgynoecious material ‘2013-12' were detected.The three genes were found in roots,stems,leaves,female flowers and male flowers.The expression of CmAP2-2 was highest in female flowers.The expression patterns of CmAP2-2 in different flower parts and different developmental stages of pistils and stamens and expression patterns of different hormone treatments were detected.It was found that CmAP2-2 gene accumulation increased along with ovary development,and CmAP2-2 after ethephon treatment.A large number was expressed of CmAP2-2 gene,indicating that the CmAP2-2 gene is involved in the development of the ovary and participates in the ethylene signaling pathway.The expression pattern of CmAP2-2 gene in ovules at 1,3,5 and 7 days after pollination was further examined,indicating that the expression level of CmAP2-2 gene gradually increased along with the development of embryos.The result of ACC and IAA treated indicated that the CmAP2-2 gene not only participated in ethylene signal pathway but also may participated in the response of auxin signal pathway.1682 bp full-length cDNA sequence of the CmAP2-2 gene from C.maxima was cloned,and the bioinformatics analysis of the protein encoded by CmAP2-2 gene was performed.