Abstract:

The saline-alkali soil is a major factor limiting the introduction and popularization of Cinnamomun bodinieri. Proline is one of the most important compatible solutes that plants accumulate for osmotic adjustment in response to saline-alkali stress. Therefore, it is of great significance to identify the key transcription factors that regulate the biosynthesis of proline and illustrate the underlying molecular mechanisms. In the present study, the seedlings were treated with 0 and 10 mmol/L Na₂CO₃ solution on the basis of hydroponic culture, respectively. Total RNA was extracted from the root tissues at 6 and 48 h after treatment to construct the yeast cDNA library induced by saline-alkali stress. The promoter sequence of CbP5CS was cloned using total DNA of C. bodinieri root system as template. The titer of the library was 4.88×10⁷CFU/mL, and the total clone number was 9.76×10⁷ CFU. The average size of the inserts was 1 000 bp, and the recombination efficiency was 100%, which met the requirements of yeast hybridization test. The length of the cloned promoter of CbP5CS was 2 012 bp, and the decoy plasmid pHIS2-CbP5CS was constructed successfully. Then the promoter region of CbP5CS was cloned and the CbP5CS promoter interacting proteins was screened by yeast one hybrid and high-throughput sequencing. The 31 unique CbP5CS-interatcting ESTs were identified from the library by co-transformation, 32 EST sequences were interacted with CbP5CS verified via yeast rotation. Six of them were annotated as transcription factors, including GW020491（zinc finger CCCH domain-containing protein 25 isoform X1）, GW028183（AtbHLH104）, GW000650（transcription factor TFIIIC）, GW007525（RING/FYVE/PHD zinc finger superfamily protein）, GW015686（GATA type zinc finger transcription factor family protein）, GW027120（AtbHLH96）. These results provide a basis for further study on the molecular mechanism of plant proline metabolism in response to saline-alkali stress.

Key words: saline-alkali stress, yeast cDNA library, CbP5CS, Cinnamomun bodinieri, upstream regulators, transcription factor