Construction Of Plant Expression Vector Of Gastrodia Antifungal Protein Gene And Transferred Into Pepper

Objective: To introduce Gastrodia antifungal protein (GAFP) gene into "ZunJiao No.1" pepper. Methods: The cDNA sequence of GAFP was cloned by RT-PCR. Plant expression vector was constructed and introduced into "ZunJiao No.1" pepper by genetic engineering technique. Results: (1) The cDNA sequence of GAFP was amplified by RT-PCR, constructing a plant expression vector -pBI121-GAFP. (2) A regeneration system of "ZunJiao No.1" pepper plant was established, optimizing the media for the stage of cotyledons differentiation and adventitious buds elongation. The optimal medium for the stage of cotyledons differentiation was comprised of MS, LC 1.0 mg/L, IAA 0.1 mg/L, ABA 0.3 mg/L, Spm100μmol/L, AgNO₃ 5mg/L and PD 6ml/L (pH 5.8～6.0); The optimal medium for the stage of adventitious buds elongation was comprised of MS, LC 0.1 mg/L, IAA 0.2 mg/L, GA₃1.0 mg/L, Spm 100μmol/L, AgNO₃ 5mg/L and PD 6ml/L(pH 5.8～6.0). The optimal medium for the stage of plantlets rooting was comprised of 1/2MS, IAA1.0mg/L and NAA 0.5mg/L(pH 5.8～6.0). (3) An efficient system of Agrobacterium tumefacient mediated pepper transformation was established. The optimal conditions for transformation of cotyledons were pre-cultured for two days, mixed with cell suspension of Agrobacterium tumefacient for inoculation in 7 minutes,co-cultivated with Agrobacterium tumefacient on the differentiation medium for two days. (4) GAFP gene was introduced into "ZunJiao No.1 "pepper. After PCR analysis of pepper plantlets It was confirmed that GAFP gene was integrated into the "ZunJiao No.1" pepper genome by the method of Agrobacterium tumefacient mediated transformation. The transgenetic "ZunJiao No.1" pepper plants were obtained. Conclusion:The vector carried a GAFP gene and contained nptll gene (a anti- Kanamycin gene) was used to transform the explants of pepper via Agrobacterium transformation. Through stringent selection of the infected explants on the medium added with Kanamycin and following PCR assays, the transgenic pepper plants were regenerated from the Kanamycin resistant explants.