Mining And Expression Analysising Of Key Gene For Flower Spot Patterning In Phalaenopsis Aphrodite Rchb.F.

Flower patterning of plants have great influence on the ornamental value of garden plants.Presently,the study of anthocyanin biosynthesis pathway has been fully understood,but there are still rare researches about the principle of the flower blotch formation.Therefore,the study on the molecular mechanism of flower spot formation is very important to reveal the subtle regulation of flower pigment patterning.Phalaenopsis amabilis belongs to Orchidaceae.It’s very popular for the beautiful and colorful flower、unique habitats in recent years.In the cultivar ’Panda’of Phalaenopsis aphrodite Rchb.F.having white flower with black spot on sepals and petals,the anthocyanins accumulate on the base of sepals and petals,which make this cultivar as a good research materials for the spot patterning in the monocotyledons.In this study,’Panda’ of Phalaenopsis was used as the research materials.Firstly,the principle of the spot formation of ’Panda’ phalaenopsis was preliminarily analyzed by the observation of the anatomical structure of petals and the determination of pigment content.The transcriptome and microRNA sequencing were performed,and the fluorescence quantitative PCR was used to detect the gene expression of structure gene and regulate gene of anthocyanin metabolic.Based on the expression differences of structure gene PeANS,PeCHI,PeF3’H,PeF3H,Pe4CL and regulate gene PeMYB11,PeMYB7,the function of key regulate gene PeMYBll,PeMYB7 was verified by using VIGS technology.All these results will lay a foundation for revealing the internet work of structure genes,regulate genes and micoRNAs responding for the spot patterning of flower of ’Panda’Phalaenopsis.The main results are as follows:1、The epidermal cell morphology of the non-spotted area of sepals was same as the spotted area.The anthocyanin is distributed widely in upper epidermis cells.The totalanthocyanin content of non-spotted area and spotted area of ’Panda’were measured by the classical pH differential method.The result showed that the anthocyanin content of spotted area of petals in ’Panda’ is 1.019mg/g,and the accumulation of anthocyanin in non-spotted area was not found.So we speculated that the formation of spot area of ’Panda’ was caused by the large number of accumulation of anthocyanin in specific position.2、12 differently expressed structure genes were obtained through transcriptome sequencing.Moreover,24 differently expressed MYB genes,42 differently expressed bHLH genes and 22 differently expressed WD40 genes were also obtained.The MYB transcription factor unigene sequences of Phalaenopsis were compared with the rice and petunia by homologous analysis to indicate the possible genetic function.3.Only 2 differently expressed microRNAs were detected that may directlly influence the expression of structure genes of anthocyanin biosynthesis.Moreover,12 microRNA targeting on MYB transcription factor genes,4 microRNA acting on bHLH transcription factor genes,and 2 microRNA acting on target WD40 transcription factors genes,were also detected respectively.4.Real-time fluorescent quantitative PCR analysis were carried out for those genes,and the results showed that the expression level of key structure genes of PeANS,PeCHI,PeF3’H,PeF3H,Pe4CL2 and the key regulate genes of PeMYB11 and PeMYB7 in spot area was significantly higher than that in non-spot area.5.The recombinant virus vector PeMYB7-PTRV29 PeMYBll-PTRV2 were successfully constructed,and the VIGS method was used to silence PeMYB7 and PeMYB11.There was no color change in ’Panda’ were observed while slience gene PeMYB7 or PeMYBll.However,the white streaks appeared in some flower when double silence on PeMYB7 and PeMYBll were performed in petunia ’Violet’.