Study On Transformation Of Pepper With Cap1 Gene By The Agrobacterium-mediated Methods

Chill Pepper?Capsicum spp.?is the one of the most important vegetable crop in the world,which have important economic value.Capsaicinoids as a unique secondary metabolism in Capsicum,is not only an important component of the flavor quality,but also widely used in chemical,food,military,health care and other fields.Currently,the capsaicionids demand gaps up to 90%in the world.However,among the world the varieties of high-spicy peppers that can be used directly for capsaicionds production is limited.To data,the capsaicin biosynthetic pathway has been basically clarified,and transcription factors that controlling capsaicin biosynthesis have been continuously identified.However,few researches have attempted to use genetic engineering to transfer the transcription factor which controlling capsaicin synthesis into plants to obtain high capsaicin content cultivars.Traditional breeding method is usually time consuming,costly,and labor-intensive,even easy produce variety failure.The use of transgenic technology can not only explore the regulatory mechanism of transcription factors associated with capsaicin synthesis-related genes,but also creat new germplasms with hereditary stability,high capsaicin content,and strong stress resistance.In this study Cap1 gene were transformed into‘59'pepper inbred lines via Agrobacterium tumefaciens mediate transformationmethod.PCR,qRT-PCR were used to detect the transgenic plants and evaluate expression level of capsaicin biosynthesis key genes and Cap1 gene in T₀transformed plants.Moreover the content of capsaicin was measured in T₀ transformed plants.T₀ validate the function of Cap1 and obtain high capsaicin transformed plants.Our results are as follows:1.Optimization of genetic transformation system dependent on tissue cultureIn this study,cefotaxine concentration comparison test was carried out,the result showed that best optimization of cefotaxine concentration was 300mg L^-1.In the genetic transformation of phosphoammonium phosphate?PPT?,the optimal select media formulation as follows:[MS+5.0 mg L^-1 6-BA?6-Benzyladenine?+1.0 mg L^-1IAA?indoleacetic acid gibberellic acid?+1.0 mg L^-1GA₃?gibberellic acid?+5.0 mg L^-1AgNO₃+300 mg L^-1Cef+sucrose 3%+agar 6.5g L^-1?PH 5.8?].Best optimization of PPT?Glufosinate ammonium?concentration were 2.5mg L^-1.While kanamycin?km⁺?have seems not suitable for select pepper'59'inbred lines transformations.2.Preliminary establishment of an Agrobacterium-mediated transformation selecting on tissue cultureThe genetic transformation method independent on tissue culture have been established,that breakthrough the traditional method of genetic transformation of plant tissue culture,targeted gene were transformed into pepper plants by means of Agrobacterium mediated transformation.The key steps were as following:the flamingo-bill explants were moved to the well-illuminated outdoor after co-culture with Agrobacterium,and PPT reagent(concentration:50mg L^-1)were dipped by cotton swab to smear the shoot tip growth points of the explants?one cotyledon and meristem were removed in the seeding?.Dipped once every three days continued three times,only a few of the explants normally forming adventitious buds.3.The biosynthesis of capsaicin can be controlled by Cap1 through regulating the expression of downstream capsaicin synthesis related genesPCR analysis were performed to detected transgenic plants of the T₀ generation pepper plants,and qRT-PCR were adopted to analysis target gene and and capsaicin synthesis gene expression.The result preliminary indicated that two RNAi transgenic plants and two overexpressed transgenic plants were identified.The percentage of transgenic was 0.057%in RNAi transformed plants.The percentage of transgenic was 0.067%in over-expressed transformed plants.Forty transformed plants with RNAi-Cap1 obtained by genetic transformation method independent tissue culture were identified by PCR analysis,the results showed that about 750 bp target band could be amplified in six transformed plants and pecent of PCR positive was 15%.The capsaicin content of T₀ transgenic plants and non-transgenic platns was determined by high performance liquid chromatography?HPLC?,respectively.The results showed that the capsaicin content of the two RNAi lines was lower than that of the non-transgenic plants.On the contrary,in the two over expressed transgenic plants,the capsaicin content was higher than that of the non-transgenic plants.respectively.The capsaicin content of six T₀ PCR-positive transformed plants that independent on plant tissue culture and control platns was determined,the results showed that the capsaicin content of five transformed plants significantly lower than the control plants.It is preliminary confirmed that Cap1 as a transcriptional factor controls the expression of capsaicin synthetic-related genes.Capsaicin biosynthes can be controlled by Cap1 through regulating the expression of capsaicin synthetic genes.