Searching for cadmium resistance genes in maize through the screening of a cDNA yeast library.

LM LOIS; CL MATAYOSHI; SM GALLEGO; D MARTÍNEZ

Virtual

Congreso; LVI SAIB-XV SAMIGE Reunión Conjunta 2020. Edición on-line.; 2020

SAIB-SAMIGE

Cadmium (Cd) is one of the most phytotoxic non-essential elements found in the environment. Like other metals, it is released to the environment by human activities and gradually is accumulated in agricultural soil, from where it can be transferred to growing plants. Thus, Cd contamination represents nowadays a serious global problem. It is imperative for crop improvement to discover stress-tolerant genes. Yeast libraries expressing heterologous cDNA are considered an efficient method to identify functional genes. Herein, a preliminary search of genes responding to Cd stress were screened from a cDNA Saccharomyces cerevisiae library that was constructed from maize (Zea mays L) seedling subjected to copper and cadmium ions. Total RNA was extracted with Trizol reagent from root and cotyledon of seedlings grown for 72 h in hydroponic culture containing diluted (1/10) Hoagland solution, 25 and 50 µM CdCl2 or CuCl2, and used for the library construction. After RNA purification, a reverse transcription was carried out using SMART technology. Then, cDNA amplification by long distance PCR, column purification and ethanol-precipitation of ds cDNA were performed. Library construction took place directly in the yeast strain Y187, via in vivo homologous recombination between the cDNA and the linearized pGADT7-Rec vector using the PEG/LiAc method. The colony forming unit of the cDNA library was 4.5x107 cells/mL. The sizes of cDNA inserts ranged from around 200 to 2,000 bp. In order to determine the library screening conditions, experiments with a concentration range of 5 to 100 µM CdCl2 included in the solid SD/-Leu medium were carried out. The criteria used to set the optimal working concentration of the metal was the one that allows a survival rate of at least 10% of library and no survival for the control yeast transformed with the empty vector pGADT7 (AD) incubated at 30 °C for 72 h. Yeast survival drastically declined with higher concentrations than 10 µM CdCl2 and no significant difference were detected with lower Cd concentration respect control yeast. In a first approach, PCR colony using pGADT7 vector primers T7 and 3′AD were performed in the surviving colonies after 10 µM Cd treatment. PCR products were then analyzed using T7 sequencing primer by capillary electrophoresis sequencing technology in an ABI 3730xl DNA analyzer (Macrogen, Korea). From seven sequences processed, four were identified as elongation factor 1-alpha, protein kinase domain, triosephosphate isomerase and Rubisco large subunit. In conclusion, the low yeast survival rate to 10 µM Cd (less than 20%) reinforces the idea that Cd tolerance is mediated by multigene families. On the other hand, PCR colony was a quick, easy and cheap methodology to isolate the amplicon used for the sequence identification. The final perspective would be to corroborate by heterologous overexpression of some of the identified sequences if they contribute to the improvement of plant tolerance to Cd.