Genetic Transformation And Expression Of Transcription Factors Related To Anthocyanin Synthesis In Aft Tomato

Aft tomato was obtained by hybridization of wild tomato species L.chilense and cultivated tomato species Solanum lycopersicum,and its fruit can induce synthesis of anthocyanins under light.The anthocyanin synthesis pathway is regulated by the MBW complex formed by MYB transcription factor,bHLH transcription factor and WD40 protein,but the regulatory network of anthocyanin synthesis in different parts of tomato tissues is still not very clear.In order to explore the expression relationship between the key regulatory factors of Aft tomato anthocyanin synthesis,and their regulatory networks involved in anthocyanin synthesis in different tissues.Aft tomato as test materials,this paper establish genetic transformation system,by real-time fluorescent quantitative PCR detection anthocyanin synthesis related MYB(SlANT1,SlANT1-like,SlAN2,SlAN2-like),bHLH(SIJAFl3,SlAN1)transcription factors in the root,stem,leaf,cotyledon,hypocotyl,petals and sepals expression quantity.SlAN2p::GUS,SlAN2-likep::GUS,SlJAF13p::GUS,SlANlp::GUS and other vectors were transferred into Aft tomato by agrobacteria-transformation method,and their expression in different tissues was determined by tissue staining,and the expression patterns of transcription factors in different tissues were further analyzed.In addition,an Aft tomato CRISPR/Cas9 gene editing system was successfully established in this study,and SlAN1,a transcription factor of bHLH,was knocked out to further study the regulation of SlAN1 in anthocyanin synthesis network in different tissues of Aft tomato.The results obtained in this paper are as follows:1.Establishment of genetic transformation system of Aft tomato.The optimal genetic induction bud medium of Aft tomato was MS+Zt 0.5 mg/L+IAA 0.1 mg/L.The optimal genetic transformation system was as follows:for tomato cotyledon explant culture 1 d,with EHA105 containing the carrier pCAMBIA2301 agrobacterium infect cotyledon 10 min,dark trained 2 d,with Tim 300 mg/L and 80 mg/L Kana culture medium for screening of 20 d,after three successive transfer,60 d screening,grow seedling put it in the rooting medium,after waiting for root detection,the average conversion efficiency was 0.46%.2.Tissue specific expression analysis of transcription factors related to Aft tomato anthocyanin synthesis.The results of real-time quantitative PCR showed that the expression of MYB transcription factor SIANT1 was low in all tissues,SlANT1-like was specifically expressed in stems,SlAN2 was highly expressed in vegetative organs,and SlAN2-like was specifically expressed in skins.SlAN1 in bHLH transcription factor is highly expressed in vegetative organs,and SlJAF13 is expressed in every tissue,similar to SlAN2.3.The CRISPR/Cas9 gene editing system of Aft tomato was successfully established.With SIAN1 as the target gene,sgRNA was designed to construct the expression vector of CRISPR/Cas9-AN1.After genetic transformation,10 transgenic plants of TO generation and 5 homozygous mutant plants were obtained,which contained two types of mutations,namely,base insertion and base deletion.5 heterozygous mutant plants were obtained.The homozygous mutant surface type was that the whole plant was free of anthocyanin,and the heterozygous mutant surface type was consistent with Aft tomato.