Construction of a grapevine cDNA library under salt stress and screening for VvRBOHA promoter-interacting proteins

【Objective】Respiratory burst oxidase homolog (abbreviated as RBOH) is an enzyme complex that plays a crucial and irreplaceable role in the physiological processes of plants. Its catalytic function is restricted by the key genes of RBOHs. Meanwhile, it is closely related to the sudden increase in oxygen consumption and the accumulation of reactive oxygen species (ROS). Therefore, a grape yeast nuclear library system under salt stress conditions was constructed, aiming to deeply and comprehensively explore the molecular regulatory mechanisms by which RBOH genes mediate grapevine responses to salt stress. This study provides theoretical support for grapevine growth and development under complex environmental conditions.【Methods】Six- year- old grapevines of Kyoho were irrigated with150 mmol·L-1 NaCl solution during the berry color-changing (veraison) period. After 12 hours, samples of grapevine roots, stems, leaves, and berries were collected. Total RNA of grapes was extracted using the Trizol method, and then mRNA was isolated using the BeaverBeads™ Oligo (dT) mRNA midi Kit. Double-stranded cDNA was synthesized using the SMART cDNA Library Construction Kit. The cDNA was ligated with three-frame attB1 recombinantly, fractionated, and collected. Small fragments were removed using the Clontech CHROMA SPIN™ and TE-1000 Column Kit. The Gateway technology was employed to construct a yeast one-hybrid (Y1H) nuclear library system for the yeast library. The yeast titer of the library was determined using a hemocytometer. Y1H technology and Next-Generation Sequencing (NGS) were used to screen for interacting proteins. 3-amino-1,2,4-triazole (3-AT) was added to the amino-acid -deficient medium to carry out self-activation verification and concentration screening of the promoter. Gene Ontology annotation (Gene Ontology, GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used to conduct enrichment analysis and annotate the candidate genes. The National Center for Biotechnology Information (NCBI) database was used to conduct gene alignment and function prediction.【Results】The A260/A280 ratio of total RNA was between 1.8 and 2.2, and there were intact 28S and 18S rRNA bands. Uniformly dispersed ds cDNA bands were obtained by amplification using 3 pairs of primers through LD-PCR. The standardization analysis process was successful, meeting the requirements for cDNA library construction. The detection results of the Escherichia coli library capacity indicated that approximately 3104 clones were obtained on a single plate, and the library titer was 3.10×108 CFU·mL-1 . A yeast one-hybrid (Y1H) nuclear library system was constructed, and the volume test results showed that the yeast titer of the library was 1.4×109 CFU·μg-1 (a colony-forming unit), the insert length was 500-2000 bp, and the recombination rate was 100%. Ten colonies were randomly selected for sequencing and compared with the NCBI database. The results demonstrated that all sequences exhibited a high degree of homology with the corresponding proteins of other plants, including grapes. This indicated that the cDNA library contained genetic information from various grapevine tissues. The VvRBOHA promoter sequence with a length of 1070 bases was successfully isolated and predicted to have multiple cis-acting response element binding sites, including a variety of hormones such as abscisic acid, methyl jasmonate, salicylic acid, ethylene, salt stress-responsive elements, and MYB and W- box binding sites. Then, it was cloned and constructed with pHIS vector, which could be successfully transformed into yeast host cells and grew on the SD/-Leu/-Trp plates containing the pGADT7 prey vector, indicating non-toxicity. However, on the SD/-Leu/-Trp/-His plates, the recombinant vector exhibited the ability to autonomously activate the His reporter gene. This auto-activation phenomenon could be completely inhibited by adding 120 mmol·L-1 3-AT. 1516 candidate interaction target proteins were screened by Y1H and next- generation sequencing (NGS). The results of KEGG pathway annotation indicated that these candidate interacting proteins were mainly involved in the most highly enriched biological pathways, such as protein metabolism, protein kinases, translation, transcription factors (TFs), ribosomes, and exosome pathways. TFs and kinases with read counts greater than 100 were selected. Homologous sequence alignment analysis was carried out using the NCBI online tool, and various proteins responding to stress signals were discovered. Eight candidate proteins with high high- throughput sequencing read counts were randomly selected for cloning, followed by Y1H verification assays. The results demonstrated that all tested proteins interacted with the VvRBOHA promoter, unequivocally confirming the high efficiency of the yeast one-hybrid library in candidate protein screening.【Conclusion】A high-capacity yeast cDNA library with optimal insert sizes was successfully constructed. Additionally, the VvRBOHA promoter was isolated, containing multiple cis-acting re-sponse elements associated with abiotic stresses, such as salt stress. Y1H screening efficiently identified numerous candidate proteins, including TFs and kinases with high sequencing read counts, which may directly or indirectly bind to the VvRBOHA promoter to regulate gene expression. These findings pioneer a novel regulatory framework for grapevine responses to abiotic stress, providing potential avenues for enhancing grape stress tolerance in the future.