Overview

We integrated error-prone PCR, the phenotype of different concentration yeast under cadmium stress into our directed evolution strategy for transporter protein,OsNramp5,that transports Mn²⁺ but not Cd²⁺.

Strain

In order to carry out the experiment smoothly, we conducted our study using the cadmium-sensitive strain (Δycf1) and the Mn²⁺ channel-defected strain(Δsmf1)that are not able to carry Mn²⁺normally through gene knockout. Δycf1 is difficult to grow normally under cadmium stress, and due to the lack of the ability to transport Mn²⁺in Δsmf1, their growth capacity is usually worse than wild type.

Screening of OsNramp5-mut strains transferring less Cd²⁺

Recombinant plasmids were transformed into yeasts, and they could not grow on the solid SD-Ura media without this kind of plasmid. In the process of library construction, we got 260 positive transformants totally. Then, the single colony of each transformant was propagated in liquid SD-Ura media. Different densities of yeast were obtained by gradient dilution, to spot on SD-Ura solid media containing ²⁺. By doing so, the effect of different yeast concentration was further avoided on the final phenotypic observation.

Screening of OsNramp5-mut strains transporting Mn²⁺ normally

The mutants with reduced transport of Cd²⁺ selected in primary screening were treated as new experimental materials. They were spotted on Mn²⁺-deficient SD-Ura media with the control group (Δsmf1-pFL61 and Δ smf1-HR). The Mn²⁺in media was specifically chelated by EGTA. As for the mutant strains, Δsmf1-pFL61 could not grow well in the media while Δsmf1-HR can, because the Δsmf1 strain cannot transport Mn ²⁺normally, they already had an additional Mn²⁺OsNramp5 transporter whose ability of transporting Cd²⁺ is decreased.

Application

The problem that Cd²⁺enriched in rice do harm to humans is still unresolved, and our subject holds the promise of achieving the evolution of OsNramp5 into an ideal Mn²⁺-specific transporter protein and therefore solving the Cd²⁺ rice problem at the source. We hope that this study will accelerate the genetic breeding of low or even cadmium-free rice and promote the utilization of cadmium-contaminated land and agricultural economic development.