Team:AHUT China/Design

Pathway and Design

Design

In order to promote the production of compound Acyl phenylethyl alcohol glycosides, we have decided to transfer RgPAL1 to enhance metabolic pathway to achieve our goal of increasing metabolic production. To realize the goal, we’ve taken the following steps.

(1) Extracting mRNA from extract of radix Rehmannia glutinosa Libosch and getting cDNA by reverse transcription.

(2) Getting coding sequence of gene RgPAL1 in Rehmannia glutinosa Libosch after measuring the genetic sequence of cDNA. Then according to available coding sequence of gene RgPAL1,we have designed and expanded complete reading frame of primers RgPAL1NSF and RgPAL1NSR, and introduced restriction enzyme sites NcoⅠand SpeⅠin primers RgPAL1NSF and RgPAL1NSR.

(3) Amplifying the gene of RgPAL1 and linking T-carrier to get the gene of pMDTM19 T RgPAL1.

(4) Taking pCAMBIA1305.1 as expression vector, and using restriction enzyme digestion of Nco Ⅰand Spe Ⅰto recover gene segment of RgPAL1 after enzyme digestion of pMDTM19 T RgPAL1. Linking gene segment of RgPAL1 with linear pCAMBIA1305.1 to get plant expression vector which contains rich RgPAL1.

(5) Transferring plant expression vector containing RgPAL1 into agrobacterium tumefaciens EHA105 to get agrobacterium engineering tumefaciens of plant expression vector containing RgPAL1.

(6) Using agrobacterium engineering tumefaciens of plant expression vector containing RgPAL1 to mediate RgPAL1 and transfer leaf explants of Rehmannia glutinosa Libosch. Then producing and gaining regeneration plant of Rehmannia glutinosa Libosch in accord with the pathway of regeneration system.

(7) Using HPLC PDA to measure the content of verbascoside in regeneration plant of Rehmannia glutinosa Libosch, and to get transgenic Rehmannia glutinosa Libosch plant containing rich verbascoside after selection.