Transcriptome Sequencing Of Viola�wittrockiana Gams. And Searcing,Expression Validation For Protential Genes Related To Petal Pigment Biosythesis

Pansy,a kind of flower of Viola from the family of Violaceae,is an important and attractive ornamental plant around the world.The diameter of its flower were about two to three inches,its petals were consitituted of three petals from the inner casement and two slightly overlapping petals from surrounding.Pansy has lots of colors in petal,mainly exhibiting red,yellow,and blue.While the specie of pansy with large and stable blotch which exhibited colors or stripes,has high value for its ornamental,so called 'cat face' for its special shape.And the related researches indicated that pansy was ideal material for the study on the formation of the flower color and blotch.For better know the molecular mechanism of spot formation in pansy,we conducted a de novo transcriptome analysis of pansy 'Mengdie' on Illumina HiSeq 2000 to present pigment metabolism genes and deduce the possible biosynthesis pathway,as well as make initial clone for the key promoter.Thus,the FPNI-PCR system for cloning promoter was optimized.The specific results were as following:(1)De novo transcriptome analysisA total of 43908 unigenes were obtained from the 118747 transcripts,which has been annotated by 6 data bases.Among them 18979 unigenes were annotated by GO;8507 unigenes were classified into 25 possible functional categories;7680 unigenes annotated by blast analysis against KAAS(KEGG Automatic Annotation Server)were mapped to 242 reference canonical pathways in pansy.Meanwhile,two types of molecular markers 194781 SNPs and 4164 SSRs were identified among 43908 sequences.11931 pairs primers were designed,12 pairs of randomly selected primers have verified the usefulness.(2)Discovery of Pigment metabolism genesA total of 99 unigenes were found to be participating in flavonoid and anthocyanidin biosynthesis,flavone and flavonol biosynthesis,carotene biosynthesis and betalain biosynthesis.The anthocyanidin biosynthesis pathway and carotene biosynthesis pathway were proposed by the result of KEGG.In particular,4,5-DOPA dioxygenase extradiol(ID,comp54934_c0)was found in transcripts,which was involved in the DOPA transform of the Betalain biosynthesis.But the tyrosinase was not found,which was necessary in the transform of DOPA.These confirmed the reason why betalain couldn't be biosynthesed was absent of DOPA,and suggested that the hypothesis of anthocyanidin and betalain can't coexist in the plant.(3)Different expression analysis of key genes in different colours:The primers of Semi-Quantitative RT-PCR were designed by the sequences of structure gene in transcripts.The different expression analysis between blotch and Non-blotch were compared in 7 flowers with different colours.These combined with the previous study,indicated that DFR gene played an important role in the formation of white colour,the expression of DFR homeotic gene DFR may have an effect on the expression of pigment in pansy,and the F3'5'H,F3'H were the key genes in the formation of blotch and delphinidin biosynthesis in pansy.(4)The gDNA clone of key genes and the optimization of FPNI-PCR reaction systemThe gDNA of ANS and DFR were cloned and bioinformatically analysed.The optimization results of various factors showed that,in the first step of FPNI-PCR,the diluted DNA should be used as template with a content of 4.5 ng?10 ng in 20 ?L reaction solution.In the third step,the reaction system should contain 0.2?mol.L-1 specific primers(GSP3),0.75 ?mol.L-1 specific primers(SP2),1.0 U Taq DNA polymerase,2.0 ?L dNTP,2.0 ?L 10 � Buffer(20 mmol.L-1,Mg2+ plus),1 ?L template DNA(the secondary PCR products diluted 100-fold with dd H2O and 1 ?L of the dilution as template)and ddH2O in total 20 ?L reaction solution.In the third step,the prefect annealing temperature were 64 ? or 66 ?.