| Lycium ruthenicum Murr.belongs to Lycium genus of Solanaceae in botany.It is common in Western and Northern China.it is rich in protein,amino acids,anthocyanins,Lycium barbarum polysaccharides,vitamins and other nutrients,with the treatment of hypertension,heart disease,irregular menstruation and other effects.Among them,Lycium ruthenicum Murr.has the highest content of anthocyanins compared to other Lycium barbarum varieties.Anthocyanin is a unique secondary metabolite of many plants,which has the functions of cold resistance,drought resistance,anti-mutation,anti-tumor and anti-oxidation,and has high medicinal and nutritional value.However,there are few molecular studies on anthocyanin accumulation in Lycium ruthenicum Murr.Due to limited by the technology of transgenic tissue culture of Lycium barbarum,this study,tomato,a relative species of Lycium barbarum,was selected for functional analysis and regulation mechanism of LrAN2.Based on the LrAN2 open reading frame sequence isolated in the previous stage,this study through expression vector construction,fluorescence quantitative PCR,determination of anthocyanin content and liquid chromatography to detect and analyze genetically modified tomato.It was further verified that the LrAN2 gene has the function of regulating anthocyanin synthesis,provides a new method for the functional verification of key genes in Lycium ruthenicum,and provides a reliable theoretical basis for the identification of the black fruit phenotype of Lycium barbarum.The results of the experiment are as follows:1.Taking Lycium ruthenicum Murr.as the research object,the LrAN2 gene was successfully cloned from it.Based on Gateway technology,PJAM1502: LrAN2 plant expression vector was obtained,which was infected into wild-type tomato marker by agrobacterium infection.The transgenic plants were tested and found that there were10 tissue culture seedlings,The 5 positive plants had an open reading frame(ORF)of774bp,which was consistent with the open reading frame of the target gene.2.By observing the phenotype of the root,stem,leaf,flower,and fruit of transgenic tomato and wild-type tomato,it was found that the root of transgenic tomato showed obvious purple phenotype,and the purple was more obvious by microscope observation,while the wild-type tomato root had no purple phenotype;There was no difference in cytochrome contents between the two types of tomato;A large amount of pigmentation was found in the transgenic tomato leaf,while the wild-type tomato Chlorophyll was mainly present in tomato leaf cells;the anther of the transgenic tomato flower was purple-black,while the wild-type tomato anther was yellow,and the difference between the flower cells of the two plants under the microscope is more obvious;Observing the fruits of the two types of tomatoes,the epidermal cells are clearly visible,there is no pigmentation,and there is no difference.3.The anthocyanin content determination results showed that the anthocyanin content in the root,stem,leaf,flower,and fruit of the transgenic tomato was higher than that in the wild-type tomato.The relative content of the anthocyanin in the transgenic tomato leaf was the highest,followed by flower and root,but the accumulation of anthocyanins in transgenic tomato fruit is not much.4.The root,stem,leaf,flower and fruit of transgenic tomato was analyzed by fluorescence quantitative PCR,the results showed that the transcription level of LrAN2 gene in the leaf of transgenic tomato plants was the highest,followed by the root;because of the anthocyanin content in the leaf of transgenic tomato was the highest,and the LrAN2 gene transcription level was also the highest,so the transgenic leaf was taken for further analysis of the structural genes of the anthocyanin metabolism pathway.The expression of each structural gene in the anthocyanin metabolism pathway in the wild-type tomato leaf was used as the control group.Fluorescence quantitative PCR display the transgenic tomato ANS,CHI,F3’H,CHS,F3’5’H,DFR,F3 H and other structural genes have increased their expression levels,and there are certain differences in the expression levels of each gene.Among them,the expression of DFR gene was the highest,was 63.73512,followed by F3’5’H gene,the expression was 40.08126,the expression of ANS gene was 13.36342,the expression of F3 H gene was 11.92258,and the expression of CHS gene was 7.912475,The expression levels of CHI gene and F3’H gene were slightly lower,1.963639 and 0.733643 respectively.5.The anthocyanins in transgenic tomato and wild-type tomato were detected by liquid chromatography-mass spectrometry,and the main anthocyanins such as delphinidin,cyaniding,pelargonidin,peonidin,malvidin were determined,and the quantitative analysis was carried out by multi reaction detection mode.It was found that delphinin-3-o-glucoside and cyanidin-3-o-glucoside existed in transgenic tomato,but could not be detected in wild tomato.The relative contents of cyanidin-3-o-rutinoside,delphinin-3-o-rutinoside and pelargonin-3-o-rutinoside in transgenic tomato were much higher than those in wild tomato.This indicated that anthocyanins could be accumulated in transgenic tomato,which further verified that LrAN2 gene could promote anthocyanin synthesis. |
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