Team:GDSYZX/Design

• Pathway
• HQT
• GluD-1 Promoter
• GluD-1 Promoter+HQT
• CaMV35S Promoter
• 35S Promoter + HQT
• How We Test?
• Western Blot
• HPLC

DESIGN

In the course of our investigation, we noticed the important effect of chlorogenic acid in Honeysuckle, and found that natural chlorogenic acid synthesis pathway also exists in other plants such as rice, a daily food. However, the content of self-synthesizing chlorogenic acid in rice and other plants is low, so we designed this project to enhance the expression of chlorogenic acid in rice seeds by inserting GluD-1 promoter and HQT gene in honeysuckle.At the same time, 35S promoter was used to design the same experiment for control, reflecting the specificity of GluD-1 expression only in seeds. We hope to improve the content of chlorogenic acid in rice seeds through the above experimental design.

Pathway

There are three biosynthetic routes that have been proposed are (1) using caffeoyl-glucoside as the active intermediate for CGA synthesis; (2) synthesizing p-coumaroyl quinate by hydroxycinnamoyl-CoA transferase (HCT) and then forming CGA by p-coumarate-3′-hydroxylase (C3′H); and (3) synthesizing CGA from caffeoyl-CoA and quinate using hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT).

It is possible that route (1) is used to synthesize or recycle CGA in a subcellular level, so it could not be the primary pathway for accumulating CGA in plants. In fact, Arabidopsis thaliana, containing both active forms of HCT and C3′H, does not accumulate CGA, which makes it unlikely that route (2) can be generally exploited by plants to accumulate significant amounts of CGA . However, route (3) has been reported as an important way for plants to synthesize CGA.

HQT

The HQT gene we use is from Lonicera japonica (GeneBank: JF261014.1), which is 1320 bp in length and encodes the synthesis of HQT, a key enzyme in the chlorogenic acid synthesis pathway. Since the experimental material we used is rice protoplasts, and the codon preference of rice is different from that of honeysuckle, we optimized the HQT gene according to the codon preference of rice, and made the optimized sequence does not contain the prohibited restriction sites of iGEM's bio-brick, and then the optimized sequence was synthesized in Tianyi Huiyuan Company. Reference: Cloning and Characterization of a cDNA Coding a Hydroxycinnamoyl-CoA Quinate Hydroxycinnamoyl Transferase Involved in Chlorogenic Acid Biosynthesis in Lonicera japonica

GluD-1 Promoter

Rice glutelin GluD-1 is a member of the glutelin family, and GluD-1 is deposited in the PB-II storage structure. The rice glutelin gene (GluD-1) can be specifically expressed in rice seeds. Studies have found that the 200bp GluD-1 promoter can specifically express the GUS gene in rice seeds, and the 1200bp GluD-1 promoter Seed + GUS gene, the expression level of GUS gene in rice seeds is very good. This project hopes to specifically increase the content of chlorogenic acid in rice seeds, so we chose the GluD-1 promoter with a length of 1200bp as our promoter for expressing HQT gene specifically in rice seeds.

Reference: Kawakatsu T, Yamamoto M P, Hirose S, et al. Characterization of a new rice glutelin gene GluD-1 expressed in the starchy endosperm[J]. J. Exp. Bot., 2008,59(15):4233-4245.

GluD-1 Promoter+HQT

In order to express HQT protein specifically in rice seeds, we linked the 1200bp promoter GluD-1 and HQT gene to pUC19, and marked the end of the gene with an HA tag to construct PGluD-1::HQT- HA vector.

CaMV35S Promote

The CaMV35S is a constitutive promoter, it can initiate gene expression in all tissues. It is persistent and does not exhibit temporal and spatial specificity.

35S Promoter + HQT

Since the rice protoplasts we used are derived from the leaf sheaths of rice seedlings, theoretically PGluD-1::HQT-HA cannot be expressed in protoplasts, so we used the constitutive expression promoter 35S to construct P35S::HQT- HA vector to detect whether the HQT gene can be expressed in protoplasts. What's more the expression of the P35S::HQT-HA vector can also be used to detect whether heterologous expression of HQT can increase the content of chlorogenic acid in rice.

How We Test?

We plan to use Western Blot and HPLC (High Performance Liquid Chromatography) to verify our vision.

Western Blot

Because the vector pUC19-HA has the HA tag, we can use the Western Blot method to compare the expression levels of PGluD-1::HQT and P35S::HQT vectors in rice protoplasts. The promoter of PGluD-1::HQTis a seed-specific expression promoter ,we speculate that no HQT protein can be detected in rice protoplasts; at the same time, the promoter of P35S::HQT is a constitutive promoter, we speculate It can detect HQT protein in rice protoplasts.

HPLC

We plan to use HPLC to detect chlorogenic acid to prove that the system we designed can successfully increase the content of chlorogenic acid in rice protoplasts.