Optimization Of Solid Phase Gene Extraction Technology And Its Application In Research Of Anthocyanin Synthesis Related Gene Expression In Iris Sanguinea

In this study,We take Iris sanguinea and Iris sanguinea f.albiflora as the research materials.Firstly,Optimized the solid phase gene extraction technology for mRNA extraction introduced from abroad;Secondly,according to the transcriptional databasethe of the I.sanguinea,separating ANS,DFR and 3GT genes from petals through RT-PCR method and conducting bioinformatics analysis.Finally,applying the new solid phase gene extraction technology to the analysis of ANS,DFR and 3GT gene expression characteristics and exploring the regulation mechanism of flower color development.The main research results are as follows:(1)Domestic probe(0.16*7mm,06Cr19Ni10)that are cheaper and easier to operate instead of introduced probe(0.14*6mm,0Cr18Ni9)to apply to solid-phase gene extraction technology.Invented two utility model patents,which are a new type of glass bottle fixator(patent number:ZL201720392787.X)and a new type of shelf to place probes(patent number:ZL201720192234.4)during the optimization of the solid-phase gene extraction technology.The optimized operation process:Pretreatment of the probe:Using the appropriate amount of n-butane,acetone,and ethanol in turn to clean the probe for 15 minutes and dry at 80℃ in a drying oven.Adding the fixed proportion of(3-glycodoxypropyl)trimethoxysilane,xylenes,ethyldiisopropylamine mixture to incubate and culture for 4 hours in a drying oven at 80℃.Embedding the probe:Washing the probe with the appropriate amount of ethyl acetate for 3 times(about 5 minutes each time).After the probe is naturally dried,putting it in a PCR tube which is filled with 20μl of 3μM Oligo dt25 solution and put the PCR tube into water bath at 37℃ for 4 h.Then,washing the probe with 4-5 ml autoclaved water of 50℃ for 3 times(each time for 5min),drying the probe naturally on the shelf.Storing the probe:Putting the dried probe in a PCR tube,and putting PCR tube in a sealed bag and vacuumized,storing it at 4℃ for 21 days.Plant RNA extraction:The probe is inserted into the plant tissue material 1-2mm for 2min and then putting it in a PCR tube which is filled with appropriate amount DEPC water,water bath at 80℃ for 10min,and RNA is dissolved in DEPC water,stored at-80℃ for use.(2)IsANS1,IsDFR1,and Is3GT1 genes are cloned.Designing specific primers based on the ANS,DFR,and 3GT gene sequences which are screened from the transcriptome database of I.sanguinea,and extracting total RNA from the petal of I.sanguinea using the improved CTAB method.Obtaining ANS,DFR,and 3GT genes through RT-PCR method.The genes are respectively named as IsANS1,IsDFR1,and Is3GT1,all of them have complete open reading frames,which are 1113bp,1071bp,and 1452bp.(3)The bio informatics analysis of IsANS1 gene showed that IsANS1 gene encodes 370 amino acids and 20 amino acid species,of which Glu(glutamic),Leu(leucine),Val(valine)and Lys(lysine)are abundant,without Pyl(pyrrolysine)and Sec(selenocysteine),with a relative molecular mass of 41.44 kDa,theoretical isoelectric point of 5.65,hydrophilic protein,containing a 2-O-ketoglutaric acid and Fe(II)-dependent oxidase superfamily of 20G-FeII_Oxy functional domains,of which 2-O-ketoglutarate has an active binding site of Arg(positions 306)and Ser(positions 308),Fe(Ⅱ)ion binding sites are His(histidine)(positions 240 and 296)and Asp(aspartic)(positions 242);the amino acid sequence encoded by IsDFR1 is 90%homologous to Iris x hollandica,and the homology is also between 71%and 80%with Muscari armeniacuM,LiliuMspeciosuM,Strelitzia reginae,Tulipa fosteriana.(4)The bio informatics analysis of IsDFR1 gene showed that IsDFR1 gene encodes 356 amino acids and 20 amino acid species,of which Val,Ala(Alanine),Glu and Leu are abundant,without Pyl and Sec,with a relative molecular mass of 39.49 kDa,theoretical isoelectric point of 5.72,hydrophilic protein.There is a NADPH binding site contents of 21-amino acid at its N-terminus and a 26-amino acid residue substrate-specific binding region which belongs to the NADB-rossmann superfamily,its substrate specific binding site is Asp at position 130 of the amino acid sequence,belonging to the Asp type,its catalytic bottom substance is dihydromyricetin(DHM);the amino acid sequence encoded by IsDFR1 is 90%and 82%homologous to I.x hollandica and Freesia hybrid cultivar,respectively,the homology is between 72%and 77%with Lilium speciosum,Curcuma alismatifolia,Tulipa fosteriana.(5)The bio informatics analysis of Is3GT1 gene showed that Is3GT1 gene encodes 483 amino acids and 20 amino acid species,of which Ala,Val,Leu,Ser and Glu are abundant,without Pyl and Sec,with a relative molecular mass of 52.69 kDa,theoretical isoelectric point of 5.61,hydrophilic protein,containing a 44-amino acid "PSPG" box at its C-terminus,belonging to the UDP-glucosyltransferase family(GTB-type),and the sugar donor is UDP-glucose;homology analysis shows that the homology of the amino acid sequence encoded by Is3GT1 gene is above 44%with Phoenix dactylifera,Musa acuminata subsp.Malaccensis,Fragaria vesca subsp.vesca.(6)The verification experiment shows that the improved SPGE technology(NSPGE)can be applied to the quantitative analysis of IsANS1,IsDFR1 and Is3GT1 gene expression.The resμlts show that The expression of IsANSI and IsDFRI genes in the leaves of I.sanguinea and I.sanguinea f.albiflora decreased gradually with the growth of leaves,Is3GT1 gene showed no significant changes with leaves growth.The expression levels of the three genes increased firstly and then decreased in I.sanguinea and I.sanguinea f.albiflora.Among them,the expression levels of IsANS1 and Is3GT1 genes were highest in the full-bloom stage and the decay period was lowest,IsDFRI gene expression was the highest in the full-bloom stage of I.sanguinea and the Initial flower period of I.sanguinea f.albiflora,the lowest level of its expression all in the decay period.The expression levels of IsANSl and IsDFR1 genes were significantly higher in the flower of I.sanguinea and I.sanguinea f.albiflora than in the leaves of the same period,and the expression levels in the flower is higher than that in the flower of the white flower stream,and the expression in the flower of I.sanguinea is higher than that in the flower of I.sanguinea f.albiflora.The expression of Is3GT1 gene is opposite,the expression level in leaves was higher than that in flowers of the same period,and the expression in the flower of I.sanguinea f.albiflora is higher than that in the flower of I.sanguinea.The results are consistent with the results of transcriptome data,which means that the IsANS1 and IsDFR1 genes are closely related to the synthesis of anthocyanins.Is3GT1 may not be involved in the anthocyanin anabolism pathway but is involved in the glycosylation modification reactions of other secondary metabolites.