Construction Of Plant Expression Vectors For As And Chr Genes, And The Study On Their Transformations In Eustoma Grandiflorum

Eustoma grandiflorum belongs to Gentianacea gentian plants. The color of their flower is elegance and bright and the shape of the flower is special and lovely. Over the past 30 years, it is one of the most popular pot and cut flower in international markets. At present, it has deep-blue,pink,rose color,yellow,white and blue color. This study attempted to express as from Antirrhinum majus and chr from Medicago trucatula in Eustoma grandiflorum (Prairie gentian 315P) by genetic engineering to change the flower color and produce a flower with yellow color at bottom part and purple color around the edge. This will provide a feasible strategy for flower molecular breeding in Eustoma grandiflorum. The results summarized as follows:1. The total RNA was extracted from yellow flower of Antirrhinum majus. The cDNA.was synthesized by reverse transcription. The coding region of as gene was amplified from the cDNA by RT-PCR and subcloned into a TA cloing vector, pMD18-T to yield pMD18-as. The pMD18-as and the gateway entry vector pENTR*-PrbcS-*T-gfp (Ma, Master thesis) were digested with restriction endonuclease and the as gene fragment t were ligated into pENTR*-PrbcS to yield an entry vector pENTR*-PrbcS-as. The PrbcS-as fragment was integrated into a Gateway plant expression vector, pK2WG7, by Gateway LR reaction to produce a light-inducible plant expression vector, pK2-35S-PrbcS-as.2. The pMD18-as and the Gateway entry vector, pENTR*-2B, were digested with restriction endonuclease, the as gene fragment were ligated into the entry vector pENTR* to yield pENTR*-as. The as gene fragment from pENTR*-as was subcloned into the plant expression vector, pK2WG7, by Gateway LR reaction to produce a constitutive plant expression vector, pK2-35S-as.3. The total RNA was extracted from yellow flower of Medicago trucatula and then reversely transcripted to cDNA. The coding region of chr gene were amplified from the cDNA by RT-PCR and subcloned into the TA cloning vector, pMD18-T, to yield pMD18-chr. The pMD18-chr and the Gateway entry vector pENTR*-PrbcS-adh (Song, Master thesis) were digested with restriction endonuclease and the chr gene fragment was ligated into pENTR*-PrbcS to yield pENTR*-PrbcS-chr entry vector. The PrbcS-chr fragment was integrated into a gateway plant expression vector, pH2WG7, by Gateway LR reaction to generate a light-inducible plant expression vector, pH2-35S-PrbcS-chr.4. The pMD18-chr and the Gateway entry vector pENTR*-2B were digested with restriction endonuclease, the chr gene fragment was ligated pENTR* to yield pENTR*-chr entry vector. The chr fragment was subcloned into the Gateway plant expression vector, pK2WG7, by Gateway LR reaction to produce a constitutive plant expression vector, pH2-35S-chr.5. The shoot induction medium for Prairie gentian 315P was optimized as MS+6-BA 1.0mg/L+IBA0.2mg/L. Its antibiotic sensitivities were also investigated. The resuslts showed that the suitable concentration of Kanamycin to screen the resistant shoots was 15mg/L. The highest concentration of Cefotaxime Na Salt (Cef) suitable to inhibit the growth of Agrobacterium was 300mg/L.6. The constructed plant expression vectors were transferred into Agrobacterium tumefaciens C58C1(pMP90).The leaf discs of Prairie gentian 315P were transformed with these plant expression vectors through Agarobacterium-mediated method.4 transgenic lines for pK2-35S-as,3 transgenic lines for pK2-35S-PrbcS-as and 2 transgenic lines for pK2-35S-chr were obtained, respectively. The transgenic plants were selectedon culture medium containing antibiotics and verified by PCR, the results indicated that as and chr gene was inserted into the genome of the Eustoma grandiflorum, respectively,The examination of as and chr expression in the transgenic plants and the observation flower color changes will be conducted in our future study.