青杄转录因子基因PwNF-YB8的克隆与功能分析

Cloning and Functional Analysis of Transcription Factor Gene PwNF-YB8 from Picea wilsonii

【目的】NUCLEAR FACTOR Y(NF-Y)转录因子在真核生物中广泛存在,通常由NF-YA、NF-YB和NFYC 3个亚基形成异源三聚体,来结合下游靶基因启动子中的CCAAT顺式作用元件,进而调控植物的生长发育进程,并参与植物非生物逆境胁迫过程。本研究对青杄中PwNF-YB8基因的表达特性及功能进行分析,揭示其参与的生理过程及对非生物逆境胁迫的响应。【方法】根据实验室前期EST测序及RNA-seq转录组数据分析结果获得青杄NF-Ys家族基因序列,克隆得到PwNF-YB8的cDNA序列,并对其编码蛋白进行序列特征和进化树分析。采用qRT-PCR分析PwNF-YB8在不同组织和花粉萌发过程中的表达特性,以及高温、盐胁迫、甘露醇、ABA处理后的表达变化。亚细胞定位揭示PwNF-YB8在细胞中发挥功能的场所。通过酵母双杂交试验、双分子荧光互补试验分别检测PwNF-YB8的转录激活活性以及与PwHAP5的互作情况。农杆菌介导花序侵染法转化野生型拟南芥(WT),获得纯合的PwNF-YB8过表达株系。利用CRISPR/Cas 9技术获得其同源基因AtNF-YB8的突变体。甘露醇和盐处理后测定野生型(WT)、空载体(VC)、突变体株系(nfyb8-cas9#1/12)和过表达株系(L4、L5)的萌发率、幼苗根长,分析比较不同株系对于渗透胁迫和盐胁迫的耐受能力。【结果】PwNF-YB8基因的开放阅读框为489 bp,编码162个氨基酸,具有典型的NF-YB保守结构域,并且与白云杉同源基因具有较近的亲缘关系。SqRT-PCR和qRT-PCR结果表明在青杄的根、茎、针叶和花粉中均能检测到PwNF-YB8的表达,其在花粉中的表达量最高。亚细胞定位试验结果显示,PwNF-YB8定位于细胞核、细胞质中。酵母自激活试验显示PwNF-YB8自身无转录激活活性。进一步对青杄幼苗进行高温(42℃)、盐胁迫、甘露醇和脱落酸处理后发现,PwNF-YB8对干旱和盐2种非生物逆境处理明显响应。PwNF-YB8参与了花粉萌发过程,在花粉萌发36 h时表达量最高。酵母双杂交和双分子荧光互补试验证明PwNF-YB8能够与NF-YC亚基中的PwHAP5互作,可能共同参与花粉管发育调控。甘露醇或盐处理下,异源过表达PwNF-YB8基因的拟南芥种子萌发率与野生型差异不显著,但其根长表现出一定的生长优势。【结论】青杄PwNF-YB8能够与PwHAP5互作,参与花粉萌发和花粉管生长过程,并在干旱、盐胁迫响应中发挥作用。

【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 c DNA 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,Na Cl 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 Na Cl 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 ℃),Na Cl 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.