Cloning And Functional Analysis Of PISTILLATA Homologous Gene From Ilex Verticillata

Ilex verticillata（L.）A.Gray（Aquifoliaceae）is a deciduous Shrub originated from North America and is widely cultivated in many countries of Asia,Europ e and America.Moreover,Winterberry Holly（I.verticillata）was introduced and widely cultivated in different areas of China for ornamental red winter fruits.Wi nterberry Holly is also a very popular ornamental cut flower or potted plant on Chinese Lunar New Year flower market.However,the male and female flower maturing or flowering at different time resulted in low fruit setting rate and orna mental value decreased.In addition,Winterberry Holly is also an interesting dioe cious shrub that an individual plant has either male or female flowers with vesti gial organs of the opposite sex.The male flower remains a vestigial pistil（pistill ode）,and the female flower remains vestigial stamens（staminodes）,which make it an excellent model for exploring female and male flower evolution of dioeciou s angiosperm.Understanding the process of floral bud differentiation and develop ment regulation mechanism of male and female tree,could help to improve the f ruit setting rate and cut-fruits ornamental value of Winterberry Holly.In Arabido psis,PI（PISTILLATA）plays a key role involved in regulating petals and stamen s development.In this study,combined with Illumina and SMRT sequencing,D GEs（differential gene expression）between male and female flower suggested tha t the significant differential expression of three PI orthologs were observed in ke y development stages in I.verticillata.Combined with paraffin sectioning techniq ue and q RT-PCR,the differential expressions of three PI-like genes were detecte d during female and male floral bud difference in winterberry holly.Furthermore,ectopic expression of Ilve PI＿1,Ilve PI＿2 and Ilve PI＿3 in Arabidopsis were perfo rmed to explore their function involved in flower development,respectively.Our data proved hence to reveal the molecular mechanism on female and male flow er development.The main results as follow:（1）Cloning and sequence alignment of Ilve PI＿1,Ilve PI＿2 and Ilve PI＿3:The DEGs during flower bud differentiation of male and female winterberry holly trees were screened by used comparing the transcriptomics.Furthermore,Differentially expressed gene transcripts with the Fold Change≥2 and FDR<0.01based on Nr annotation,Three PI（PISTILLATA）orthologous genes,Ilve PI＿1（tr anscript＿5146）,Ilve PI＿2（transcript＿5261）and Ilve PI＿3（transcript＿5403）,were fo und to be involved in regulating the flower bud morphological differentiation of male and female trees of.Three Ilve PI＿1,Ilve PI＿2 and Ilve PI＿3 genes containing complete ORF（Open Reading Frame）were isolated by RACE technique.Each of the three PI-like genes encodes a MADS transcription factor composed of 211 amino acid residues.Protein homologous alignment and molecular phylogeny tr ee grouped Ilve PI＿1,Ilve P＿2 and Ilve PI＿3 into the PI/GLO lineage of MADS-B ox gene family.In addition,the Ilve PI＿1 shows 91.94%identity to Ilve P＿2,but only 86.73%identity to Ilve PI＿3.Moreover,a highly conserved PI motif compos ed of 16 amino acid residues was lying at the C-terminal region of PI-like trans cription factor in I.verticillata.（2）Expression patterns of Ilve PI＿1,Ilve PI＿2 and Ilve PI＿3 in I.verticillata:The expressions of Ilve PI＿1,Ilve PI＿2 and Ilve PI＿3 genes were detected com bined with plant microscopy and q RT-PCR during the key stages of flower bud differentiation between male and female Holly trees,respectively.Ilve PI＿1,Ilve PI＿2 and Ilve PI＿3 were obviously expressed in the flower bud differentiation proce ss of male trees,and the expression levels increased continuously during stamen development,and reached the peak when pollen was mature.The relative express ion levels of Ilve PI＿1 and Ilve PI＿2 genes were significantly higher than those of Ilve PI＿3（LSD,p<0.01）.The relative expression levels of Ilve PI＿1,Ilve PI＿2 and Ilve PI＿3 were the highest in the early stage of female floral bud differentiation（F₁stage）,when microspore mother cells formed in anther of vestigial stamens.Moreover,the expression level of Ilve PI＿1 was significantly higher than that of Ilve PI＿2 and Ilve PI＿3 in flower bud of female trees at F₁stage（LSD,p<0.01）.The expression level of Ilve PI＿1 dropped off at F₂stage of the female flower buds.Then,the expression level of the three genes sustained at a low level until the flower buds matured in female trees.Morphological and cytological characte ristics showed that microspore mother cells began to disintegrate at F₂stage and subsequently disappeared completely at F₃stage.The anther sac is with empty chamber and thickened wall.The expressions of Ilve PI＿1,Ilve PI＿2 and Ilve PI＿3genes were positively correlated with the stamen development,especially the poll en development.（3）Functional analysis of Ilve PI＿1,Ilve PI＿2 and Ilve PI＿3 genes in I.vertici llata:Ectopic expressions of Ilve PI＿1,Ilve PI＿2 and Ilve PI＿3 in Arabidopsis showe d that 35S::Ilve PI＿1,35S::Ilve PI＿2 and 35S::Ilve PI＿3 transgenic plants separately produced obvious petalloid sepals,which indicated that three PI-like genes were involved in regulation of petal development.The 35S::Ilve PI＿2 transgenic homo zygote pi-1 Arabidopsis completely restored the petal structure in whorl 2and fila ment-like organ in whorl 3,The 35S::Ilve PI＿3 transgenic homozygote pi-1 Arabi dopsis completely restored the petal structure in whorl 2 and filament-like organ attached with stigmatic papillae in whorl 3.Moreover,expression levels of 3 PI-like genes were highest at key of stamen development in male and female tree,which suggested that three PI-like genes played key roles in regulating stamen d evelopment.Our results suggested that Ilve PI＿1,Ilve PI＿2 and Ilve PI＿3 work toge ther to specify petal and stamen identities in Ilex verticillata.