Application Of Anthocyanin In Mulberry Genetic Transformation System

Mulberry is a perennial dicotyledonous woody plant.China is the origin of mulberry planting,mulberry breeding silkworm has a history of more than 5,000 years,casting the world-famous "Silk Road".Mulberry is also an eco-economic tree species,showing strong vitality and vitality in the fields of ecological environment management and medical care.With the analysis of mulberry genome,the study of mulberry has entered the post-genomic era.However,there is still a lack of mature transgenic system of mulberry,which seriously restricts the functional study of mulberry,and has become the bottleneck of improving the quality and efficiency of mulberry industry as well as the development of modern mulberry discipline.Agrobacterium-mediated genetic transformation system requires three conditions:first,a stable genetic regeneration system;second,effective methods of infection;third,intuitive and efficient transgene screening technology.The lack of efficient and mature transgenic systems in mulberry indicates that the regeneration,infection and screening of mulberry should be further optimized.As the most important part of screening transgenic plants---efficient screening technology has been valued and used by researchers.Various reporter genes such as fluorescent proteins represented by Green Fluorescent Protein(GFP),β-glucuronidase gene(GUS),and luciferase gene have been widely used in transgenic studies.However,traditional reporter genes can only function with the help of related equipment or expensive substrates,and there are significant limitations to the screening of field or large-scale samples.In view of this,the reporter genes that are visible to the naked eye are increasingly developed and applied.The colorful compounds synthesized by plants are potentially visible reporter genes.Anthocyanin,an important water-soluble pigment widely distributed in plants,is the main color component of plant organs and has anti-oxidation and anti-inflammatory functions.Mulberry is rich in anthocyanins,and the synthesis of anthocyanins is regulated by both structural and regulatory genes in the anthocyanin synthesis pathway.Among them,R2R3-myeloblastosis(MYB),basic helix-loop-helix(b HLH)and WD40 form a MYBb HLH-WD40(MBW)ternary complex to regulates the expression of anthocyanin structural genes.In summary,this study started from screening mulberry germplasm resources,using anthocyanins as a visual reporter gene to establish a stable Agrobacterium-mediated mulberry genetic transformation system.The main results obtained are as follows:1.In 2018,39 naturally pollinated mulberry germplasm resources were collected from the ecological mulberry garden of Southwest University.The seed germination rate,seedling growth situation,hypocotyl callus induction,and callus differentiation ability were investigated.Weeping mulberry with good comprehensive performance were screened and used to construct the mulberry genetic transformation system;2.The anthocyanin content was significantly upregulated in the transgenic tobacco by overexpressing the fusion expression vector Gh PAP1,which constituted the maize leaf color gene Lc and the cotton anthocyanin regulating gene Gh PAP1.Then,GHPAP1 was transformed into the hypocotyls of mulberry by Agrobacteria-mediated genetic transformation.After culturing for about 140 days,red callus was observed,and positive GUS histochemical staining was detected in the induced resistant callus,which initially indicated that exogenous genes have been integrated into the mulberry genome and can regulate the synthesis of mulberry anthocyanins.The results of quantitative PCR(q RTPCR)indicated that the expression level of anthocyanin biosynthesis related genes in mulberry red callus was generally significantly up-regulated,and the color intensity of red callus was positively correlated with the expression level of anthocyanin biosynthesis related genes.The results of non-targeted metabolomics analysis showed that compared with the empty and wild-type controls,the pelargonium-3-O-glucoside,cyanidin-3-Oglucoside,pelargonidin-3-rutinoside,cyanidin-3-O-rutin and rutin in red callus increased significantly.3.In order to further improve the efficiency of mulberry genetic transformation,according to the red phenotype presented by the increased anthocyanin content of positive callus after transformation,the co-cultivation conditions,medium hormone ratio,transformation conditions and explant culture conditions of mulberry genetic transformation system were optimized.Finally,the cycle time required for induce the red phenotype of transgene positive callus was shortened from 140 days to 30 days,and the transgene positive rate was increased from 13.3% to 37.0%,improved the efficiency of the visual tracking expression system using anthocyanin as reporter,and established an efficient and stable mulberry genetic transformation system.The anthocyanin visual screening strategy adopted in this study facilitates accurate,rapid,and large-scale screening of mulberry transgenic positive materials,and improves the detection efficiency of transgenic mulberry.It lays the foundation for establishing a mature,stable and efficient transgenic platform in mulberry trees,which is important for functional analyses of mulberry genes.