Part:BBa_K1751002

Background and priniciples

The gene RgPAL1 can express Phenylalanine ammonia lyase（PAL）, which is an enzyme that catalyzes a reaction converting L-phenylalanine to ammonia and trans-cinnamic acid.PAL is the first and committed step in the phenyl propanoid pathway and is therefore involved in the biosynthesis of the polyphenol compounds. In our project, PAL plays an important role in L-phenylalanine’s deaminization to produce trans-cinnamic acid.

Phenylalanine ammonia lyase (PAL) is an enzyme that catalyzes a reaction converting L-phenylalanine toammonia and trans-cinnamic acid. Phenylalanine ammonia lyase (PAL) is the first and committed step in the phenyl propanoid pathway and is therefore involved in the biosynthesis of the polyphenol compounds such as flavonoids,phenylpropanoids, and lignin in plants. Phenylalanine ammonia lyase is found widely in plants, as well as someyeast and fungi, with isoenzymes existing within many different species. It has a molecular mass in the range of 270-330 kDa. The activity of PAL is induced dramatically in response to various stimuli such as tissue wounding,pathogenic attack, light, low temperatures, and hormones. PAL has recently been studied for possible therapeutic benefits in humans afflicted with phenylketonuria. It has also been used in the generation of L-phenylalanine as precursor of the sweetener aspartame.

The enzyme is a member of the ammonia lyase family, which cleaves carbon-nitrogen bonds. Like other lyases, phenylalanine requires only one substrate for the forward reaction, but two for the reverse. It is thought to be mechanistically similar to the related enzyme histidine ammonia-lyase (HAL). The systematic name of this enzyme class is L-phenylalanine ammonia-lyase (trans-cinnamate-forming). Previously, it was designated PAL, but that class has been redesignated as phenylalanine ammonia-lyases, tyrosine ammonia-lyases, and phenylalanine/tyrosine ammonia-lyases. Other names in common use include tyrase,phenylalanine deaminase, tyrosine ammonia-lyase, L-tyrosine ammonia-lyase, phenylalanine ammonium-lyase, PAL, andL-phenylalanine ammonia-lyase.

Enzyme Mechanism

Phenylalanine ammonia lyase is specific for L-Phe, and to a lesser extent, L-Tyrosine. The reaction catalyzed by PAL is the spontaneous, non-oxidative deamination of L-phenylalanine to yield trans-cinnamic acid and ammonia.

L-phenylalanine --> trans-cinnamate + NH3

The cofactor 3,5-dihydro-5-methyldiene-4H-imidazol-4-one (MIO) is involved in the reaction and sits atop the positive pole of three polar helices in the active site, which helps to increase its electrophilicity.MIO is reported to attack the aromatic ring of L-Phe in a Friedel-Crafts-type reaction, which activates the C-H bond and leads to cleavage of the bond. The carbanionintermediate formed by this mechanism is stabilized by partial positive regions in the active site. The mechanism of the reaction of PAL is thought to be similar to the mechanism of the related enzyme histidine ammonia lyase.PAL is inhibited by trans-cinnamic acid, and, in some species, may be inhibited trans-cinnamic acid derivatives.D-Phe and D-Tyr are competitive inhibitors.

Design

Pathway: Phenylalanine ammonia-lyase，PAL can catalyze L-phenylalanine’s deaminization to produce cinnamate. It serves as the enzyme that links primary metabolism, phenylaprapanoid metabolism and catalytic phenylaprapanoid metabolism in the first phase. It is also the key and rate-limiting enzyme in metabolic pathway of phenylalanine.

PAL plays an important role in the pathway of producing compound Acyl phenylethyl alcohol glycosides from Rehmannia glutinosa Libosch and in the process of producing cinnamate by phenylalanine’s deaminization. It has greatly limited the expression of the whole synthetic pathway. Besides, by inducing the high expression of PAL, it has made a large amount of cinnamate from phenylalanine be involved in the next synthetic pathway to increase the expression of compound Acyl phenylethyl alcohol glycosides.

In Rehmannia glutinosa Libosch, the compound of APeGs can synthesize the key compound and can be available by being combined in the pathways of phenylalanine and tyrosine respectively with chorismate through the precursor way. In the pathway of phenylalanine, phenylalanine ammonia-lyase serves as a very important key enzyme in the procedure of Phenylalanine’s turning into cinnamate. It can catalyze phenylalanine’s deaminization to produce cinnamate, and then involve in the later procedures of synthesis.

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.

Results

Ⅰ. Verification of Carrier Construction

Fig.1. Electropherogram of RNA Extract’s Result

After getting the extract of Rehmannia glutinosa Libosch leaves, total RNA can be available through separation techniques. The quality of eluted RNA can be identified by electrophoresis in modified formaldehyde glue.

Fig.2. Electropherogram of PCR Testing Result

After the availability of RNA and reverse transcription of cDNA, we have amplified the target gene we need. With the knowledge of the length of target gene, we’ve verified its conformance to length requirements through electrophoresis.

Fig.3. Electropherogram of Digestion Veritication Result

In order to construct the expression carrier, we’ve conducted enzyme digestion of pCAMBIA1305.1 carrier and cloning carrier of target gene at the same time, to get carrier backbone of pCAMBIA1305.1 and gene segments of RgPAL1. We have got two segments meeting the length requirement through electrophoresis verification.

Ⅱ. Verification of Transformation

Fig.4. Electropherogram of PCR Veritication Result

After the construction of expression carrier, we have mediated gene RgPAL1 through agrobacterium engineering tumefaciens to transform explant of Rehmannia glutinosa Libosch leaves. In order to ensure target gene’s existence in the agrobacterium, we have verified through electrophoresis that the agrobacterium can generate gene RgPAL1 which can meet the length requirement.

Fig.5.A. HPLC-PDA spectrum of verbascoside standards

Fig.5. B. HPLC-PDA spectrum of untransformed and regulate Rehmannia glutinosa Libosch extract

Fig.5. C. HPLC-PDA spectrum of extract of Rehmannia glutinosa Libosch of transformed RgPAL1

After the agrobacterium has been transformed and the plant has been cultivated for a while, we determinated the content of verbascoside in transgenic Rehmannia glutinosa Libosch by HPLC-PDA, to verify that the plants containing the target gene can highly express specified products.

The specific steps are as follows.

Preparing standard solutions: 50 mg of verbascoside standards are weighted and taken to 50 mL with methanol of 50% mass concentration. Then standard solutions of 2.5, 5, 10 and 100 µg·mL -1 are diluted respectively for standby;

Drawing a standard curve chart: Testing verbascoside standard solutions of different concentrations prepared in step one by HPLC-PDA. Drawing a standard curve chart of peak area Y and standards content X with linear equation of Y = 34731X - 9296.6，R 2 =0.9997 with µg as the unit of standards content. Linear regression analysis shows that the concentration of verbascoside is between 2.5 µg·mL -1 and 100 µg·mL -1 with a good linear relationship between peak area Y and standards content X;

Determinating the content of verbascoside in Rehmannia glutinosa Libosch: Taking the transgenic Rehmannia glutinosa Libosch after selection and cultivation into the oven of 40℃. Dry it into constant weight and ground into powder. Selecting the powder with 0.45mm sieve and extracting 1g powder after sieving with 18 mL methanol ultrasonic of 50% mass concentration. After that, taking 25mL of the supernatant and measuring the peak area of verbascoside with HPLC-PDA. The linear equation in step two can be used to calculate the content of verbascoside.

The result proves that the content of verbascoside in normal non-transgenic Rehmannia glutinosa Libosch is 1.27 mg·g -1, while in transgenic Rehmannia glutinosa Libosch of RgPAL1, the content reaches 3.27 mg·g -1, which is 2.57 times of that in non-transgenic Rehmannia glutinosa Libosch.

Useful Resources

（1）https://en.wikipedia.org/wiki/Phenylalanine_ammonia-lyase