Difference between revisions of "Part:BBa K3458002"

Latest revision as of 03:46, 17 August 2020

GluD-1 Promoter

The promoter of the Oryza sativa L. glutelin gene (GluD-1), due to the deviation in the GCN4 motif, this pomoter has the characteristics of being specifically expressed in the endosperm of Oryza sativa L..

Usage and Biology

Rice grain provides a major source of carbohydrates and proteins, especially in developing countries. Starch and storage proteins are predominantly deposited in the rice grain endosperm during seed maturation, and are used as a source of nitrogen and carbon for the germinating seedling.

In rice, glutelin is the major seed storage proteins. Glutelin and globulin are deposited in protein body II (PB-II) storage vacuoles. A number of biopharmaceuticals, nutriceuticals containing health-promoting peptides, and mineral binding peptides have been produced using rice endosperm as a bioreactor. Moreover, transgenes can be highly expressed in a specifific tissue during a specifific developmental stage by employing native SSP promoters.

Oryza sativa L. glutelin (GluD-1) is a member of the glutelin family. The expression of GluD-1 gene has unique temporal and spatial characteristics. GluD-1 was predominantly expressed in the inner starchy endosperm beginning about 5 DAF and steadily increased until maturity at 30 DAF. Its spatial specificity is caused by the characteristics of the GluD-1 promoter. Studies have shown that Only about 0.2 kb of the GluD-1 promoter was suffificient to confer inner starchy endosperm-specifific expression.

Fig. 1 Truncation analysis of the GluD-1 promoter

Studies have found that the 200bp GluD-1 promoter can specifically express the GUS gene in rice seeds, and the 1200bp GluD-1 promoter + GUS gene, GUS gene expression level 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.

Characterization

The GluD-1 promoter cannot express the gene in rice protoplasts due to its specific expression in Oryza sativa L. endosperm. To verify this, we designed two composite parts and used western blot to compare GluD-1 promoter and 35S constitutive promoter).（more information on BBa_K3458003 and BBa_K3458004)

Fig. 2 The result of the Western Blot

The results of western blot showed that the 35s promoter can initiate the expression of HQT gene in Oryza sativa L. protoplasts while the GluD-1 promoter cannot.This result can prove the specificity of GluD-1 promoter expression.

References

[1]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.

[2]Ye X, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, Potrykus I. 2000. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287, 303–305.

[3]Paine JA, Shipton CA, Chaggar S, et al. 2005. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature Biotechnolgy 23, 482–487.

[4]Qu LQ, Takaiwa F. 2004. Evaluation of tissue specifificity andexpression strength of rice seed component gene promoters intransgenic rice. Plant Biotechnology Journal 2, 113–125.

Sequence and Features