Construction And Genetic Transformation Of RNAi Plant Expression Vector In Self-incompatibility SFBB Gene From Korla Fragrant Pear

Object: Korla Fragrant Pear(Pyrus sinkiangensis Yü)is a unique and famous cultivar in the Xinjiang Uyghur Autonomous Region.Self-incompatibility which exhibits in Korla fragrant pear has restricted the development of its industrialization.The objective of the study is to establish the stable and effective regeneration system of leaves in-vitro of Korla Fragrant Pear seedlings.To lay a foundation for self-incompatibility improvement and self-compatibility pear cultivars breeding by plant gene engineering technology.Methods: 1、The aseptic seedling leaves sub-cultured for 30 days of Korla fragrant pear cultured in vitro were used as material.Firstly,1/2 MS was used as the basic medium,supplemented with the plant growth regulator thidiazuron(TDZ 1.0 mg/L)and Indolebutyric acid(IBA 0.3-0.9 mg/L)orα-Naphthaleneacetic acid(NAA 0.3-0.9 mg/L)combined respectively in different formulas.The leaves were cut into 0.5cm2 for 21-day dark cultured with their abaxial surface on the medium.Each formula was inoculated with 30 leaves and repeated 3 times.By comparing the callus induction frequency and regeneration of adventitious buds,the growth regulator for dedifferentiation formation of leaves and dedifferentiate into callus tissues was optimized in 14 kinds of formulas.Then Ag NO3(0,0.5,1.0 mg/L)of different mass concentration was added into the optimum medium formula of regeneration of adventitious buds.Finally,When the shoots were about 2 cm in height,they were transferred to the root medium on 1/2 MS medium supplemented with IBA(0.5 mg/L)and NAA(0.3,0.6,0.7,0.9,1.1,1.4 mg/L)combined respectively in different formulas.Each formula was inoculated with 30 the aseptic seedlings and repeated 3 times.After 7-day culture in the dark,the most appropriate root induction medium could be screened out by comparing the rooting rate,average root-shoot ratio and the average root length.2、One full-length,pollen-specific SFBB-γ(SFBBx-gamma(Gen Bank 登录号:KU756268))c DNA gene was cloned from Korla Fragrant Pear by our experiment.This c DNA gene included an F-box region and four variable regions(V1,V2,V3,and V4).Its length was 1344 bp and it contained one complete 1191 bp Open Reading Frame which encoded 396 amino acids.The RNAi target sites was F-box region and variable region V3 fragment of SFBB,a 242 bp exogenous fragment was the loop of hairpin from the genomic DNA fragment of cotton,the ihp RNA was obtained by fusion PCR inserted into the expression vector.The recombinant plasmids were transferred into GV3101 by eletroporation.3 、 The RNAi vector was introduced into Korla Fragrant Pear seedlings by leaf disk transformation method.Histochemical staining of GUS was used for identification of transgenic plants.The RNAi expression vector were transferred into Korla Fragrant Pear by pollen-tube pathway,the fruit set rate were analyzed by significant differences.Results: 1、The results indicated that the optimal medium for adventitious shoot induction was 1/2 MS supplemented with 1.0 mg /L TDZ,0.5 mg /L IBA and 0.5 mg /L Ag NO3,the frequency of callus formation and adventitious shoot regeneration were 100 % and 84.92%,respectively.The appropriate rooting induction medium was 1/2 MS + 0.5 mg /L IBA + 0.9 mg /L NAA,the rooting rate could reach 100% after 7-day culture in the dark.The regeneration system of leaves in vitro of Korla Fragrant Pear seedlings was established,which laid foundation for genetic transformation of pear.2、The RNAi expression vectors p CAMBIA1304-RNAi-SFBB-F-box and p CAMBIA1304-RNAi-SFBB-V3 were constructed successfully and transferred into GV3101.3 、 The results of histochemical staining of GUS showed that target gene(SFBB-F-box or SFBB-V3)were integrated into Korla Fragrant Pear seedlings.Transformed by RNAi expression vector stigma-dropping,fruit set rate of self-pollination in Korla Fragrant Pear improved significantly.Both of RNAi expression vectors targeted self-incompatibility gene caused silence of gene and the failure of self-incompatibility reaction.Conclusion: The regeneration system of leaves in vitro of Korla Fragrant Pear seedlings was established.The RNAi expression vectors targeted self-incompatibility SFBB-F-box and SFBB-V3 domain in Korla Fragrant Pear were successfully constructed,and transformed into leaves in vitro culture by leaf-disk transformation method,and into ovary by pollen-tube pathway,respectively.GUS staining were obtained successfully.These results laid the foundation for the genetic transformation of RNAi expression vector in self-incompatibility gene,which has important value for the breeding of self-compatible pear cultivars and other Rosaceae plants.