关键词: 青杄, NF-Y转录因子, 基因表达, 胁迫响应, 花粉萌发

Abstract:

Objective: NUCLEAR FACTOR Y(NF-Y) is a ubiquitous transcription factor in eukaryotes. It consists of three subunits, NF-YA, NF-YB and NF-YC, all of which can form heterotrimer to regulate the expression of downstream genes by binding to cis-acting elements of CCAAT in the promoter region, thus participating in plant development and abiotic stress response. In this study, the expression characteristics and functional analysis of the PwNF-YB8 gene in Picea wilsonii were analyzed in order to reveal the physiological processes involved and the responses to abiotic stress. Method: According to the sequences of P. wilsonii NF-Ys family genes obtained from the pre-laboratory EST sequencing and RNA-seq transcriptome database, the cDNA sequence of PwNF-YB8 was cloned and its encoded protein was analyzed by sequence characteristics and evolutionary tree. qRT-PCR was used to analyze the expression characteristics of PwNF-YB8 in different tissues and pollen germination process, as well as the expression changes under heat, NaCl stress, mannitol treatment, ABA treatment. Subcellular localization revealed where PwNF-YB8 worked in cells. The transcriptional activation activity of PwNF-YB8 and its interaction with PwHAP5(NF-YC subunit) were detected by yeast two-hybrid assay and bimolecular fluorescence complementation assay, respectively. The Arabidopsis Col-0(WT) was transferred by floral dip method to obtain homozygous PwNF-YB8 overexpressing lines. Mutants of its homologous gene AtNF-YB8 were obtained by using CRISPR/Cas 9 technology. The germination rate and seedling root length of wild type (WT), empty vector (VC), mutant lines (nfyb8-cas9#1/12) and overexpression lines (L4, L5) were measured under mannitol and NaCl treatment, and the tolerance of different lines to osmotic stress and salt stress was analyzed and compared. Result: The open reading frame of PwNF-YB8 gene is 489 bp, which encodes 162 amino acids, has a typical NF-YB conserved domain, and has a close relationship with the homologous genes of Picea glauca. The SqRT-PCR and qRT-PCR both showed that PwNF-YB8 was expressed in roots, stems, needles and pollen of P. wilsonii, with the highest expression in pollen. Subcellular localization showed that PwNF-YB8 was localized in the nucleus and cytoplasm. Yeast two-hybrid assay showed that PwNF-YB8 had no transcriptional activation activity. After further treatments of the seedlings of P. wilsonii with heat (42℃), NaCl stress, mannitol and ABA, it was found that PwNF-YB8 responded significantly to abiotic stresses such as drought and salt. PwNF-YB8 participates in the pollen germination process, and its expression is the highest at 36 h of pollen germination. The yeast two-hybrid experiment and bimolecular fluorescent complementary experiment proved that PwNF-YB8 can interact with PwHAP5, and may participate in the regulation of pollen tube development. Under mannitol or salt treatment, the seed germination rate of Arabidopsis overexpressing the PwNF-YB8 gene was not significantly different from that of the wild type, but its root length showed a certain growth advantage. Conclusion: Picea wilsonii PwNF-YB8 gene can interact with PwHAP5, participate in pollen germination and pollen tube growth, and play a role in response to drought and salt stress.

Key words: Picea wilsonii, NF-Y transcription factor, gene expression, stress response, pollen germination