Identification of PtNF-YC1 of Pinus tabuliformis and its molecular mechanism involved in regulation of cone development
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摘要:
目的 针叶树中NF-Y核因子调控球花发育的研究尚未见报导，对油松PtNF-YC1基因克隆、表达特性及功能分析，旨在为针叶树NF-Y基因家族在球花生殖发育中的功能研究提供依据。 方法 （1）利用系统进化树分析油松PtNF-YC1与拟南芥NF-YC亚家族蛋白的亲缘关系；（2）瞬时转化烟草检测PtNF-YC1的亚细胞定位；（3）根据转录组数据分析PtNF-YC1在油松不同组织中的表达特性；（4）PtNF-YC1异源转化拟南芥，分别比较长日照和短日照条件下转基因拟南芥不同株系的开花时间，并对长日照下各株系进行转录组测序，筛选响应PtNF-YC1调控开花的相关基因；（5）通过Y2H和BiFC验证PtNF-YC1与候选蛋白之间互作。 结果 PtNF-YC1基因的开放阅读框为897 bp，编码299个氨基酸，含有典型NF-YC保守结构域，与AtNF-YC3/4/9具有较高同源性。亚细胞定位结果显示，PtNF-YC1定位在细胞核和细胞质。PtNF-YC1在针叶、营养芽、雌雄花芽和根中都能表达，但在雄球花中表达丰度最高。PtNF-YC1异源转化拟南芥可推迟其短日照下开花时间。Y2H和BiFC证明PtNF-YC1与PtCOL5存在相互作用。 结论 PtNF-YC1可调控成花时间，是油松光周期途径诱导球花发育的候选基因。 Abstract:Objective The research on the regulation of NF-Y nuclear factor on cone development in conifers has not been reported yet. Through the cloning, expression characteristics and functional analysis of PtNF-YC1 gene of P. tabuliformis, it provides a basis for the functional study of conifer NF-Y gene family in the reproductive development of conifers. Method (1) The relationship between PtNF-YC1 and Arabidopsis thaliana NF-YC subfamily proteins was analyzed by phylogenetic tree. (2) Tobacco was transiently transformed to detect the subcellular localization of PtNF-YC1. (3) The expression characteristics of PtNF-YC1 in different tissues of P. tabuliformis were analyzed based on transcriptome data. (4) PtNF-YC1 was heterologously transformed into Arabidopsis, and the flowering time of different transgenic Arabidopsis lines under long-day and short-day was compared. The transcriptome sequencing of each transgenic line under long-day was performed to screen the genes related to PtNF-YC1 regulating flowering. (5) The protein interaction between PtNF-YC1 and candidate proteins was verified by Y2H and BiFC. Result The open reading frame of PtNF-YC1 was 897 bp, which encoded 299 amino acids, had a typical NF-YC conserved domain, and a close relationship with the homologous genes of AtNF-YC3/4/9. The subcellular localization showed that PtNF-YC1 was localized in the nucleus and cytoplasm. The analysis of expression patterns in different tissues showed that PtNF-YC1 could be expressed in needles, vegetative buds, male and female cones and roots, but the expression abundance was the highest in male cones and stems. PtNF-YC1 heterologous transformation of Arabidopsis delayed flowering under short day. It is proved that PtNF-YC1 interacts with PtCOL5 through Y2H and BiFC. Conclusion PtNF-YC1 has the function of regulating flowering time and is a candidate gene for inducing cone development through photoperiodic pathway of P. tabuliformis. -
Key words:
- Pinus tabuliformis /
- PtNF-YC1 /
- photoperiod regulation /
- cone development
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图 1 油松PtNF-YC1与拟南芥和小鼠NF-YC蛋白序列比对
Figure 1. Alignment of P. tabuliformis PtNF-YC1 with Arabidopsis and mouse NF-YC protein sequences
图 2 油松PtNF-YC1与拟南芥AtNF-YCs蛋白系统发育分析
Figure 2. Phylogenetic analysis of PtNF-YC1 protein from P. tabuliformis and AtNF-YCs protein from A. thaliana
图 3 PtNF-YC1蛋白三级结构预测（a）和亚细胞定位（b）
Figure 3. Protein tertiary structure prediction (a) and subcellular localization (b) of PtNF-YC1
图 5 长日照（a）和短日照（b）条件下野生型Col-0和35S::PtNF-YC1过表达株系的表型
Figure 5. Phenotypes of wild-type Col-0, 35S::PtNF-YC1 overexpressing lines under long-day (a) and short-day conditions (b)
图 6 油松PtNF-YC1与PtCOL5酵母双杂交互作（a）和双分子荧光互补（b）验证结果
SD/-Leu-Trp和SD/-Leu-Trp-His-Ade培养基用于检测互作验证，AD-T/BD-p53（PC）和AD-T/BD-PtNF-YC1（NC）为阳性对照和阴性对照。SD/Leu Trp and SD/Leu Trp His-Ade media are used for detection of interaction validation, with AD-T/BD-p53 (PC) and AD-T/BD-PtNF-YC1 (NC) as positive and negative controls.
Figure 6. Yeast two-hybrid interaction (a) and bimolecular fluorescence complementation (b) of PtNF-YC1 and PtCOL5 of P. tabuliformis
表 1 PtNF-YC1引物信息
Table 1. Primer information of PtNF-YC1
引物名称 Primer name 引物序列（5′—3′） Primer sequence (5′−3′) 用途 Usage PtNF-YC1-F ATGGACCACCACAACCACCAC 基因克隆
Gene clonePtNF-YC1-R GTTGGCAGAACGAGGGGGAG pSPYNE-PtNF-YC1-F TTAACCGGGCTCAGGCCTATGGACCACCACAACCACCAC 双分子荧光互补
Bimolecular fluorescence complementationpSPYNE-PtNF-YC1-R GAGCGGTACCCTCGAGGTTGGCAGAACGAGGGGGAG pBI121-eGFP-PtNF-YC1-F CACGGGGGACTCTAGAATGGACCACCACAACCACCA 亚细胞定位
Subcellular localizationpBI121-eGFP-PtNF-YC1-R CCATGGTACCCCCGGGGTTGGCAGAACGAGGGGGA BD-PtNF-YC1-F GGAGGCCGAATTCCCGGGGATGGACCACCACAACCACC 酵母双杂交
Yeast two hybrid systemBD-PtNF-YC1-R GCCGCTGCAGGTCGACTCAGTTGGCAGAACGAGGGG AD-PtCOL5-F CAGTGAATTCCACCCGGGGATGGTGAAGGAAGAAGACAAG 酵母双杂交
Yeast two hybrid systemAD-PtCOL5-R TCATCTGCAGCTCGAGTCAATAAGATGGAACAACTCCAT pSPYCE-PtCOL5-F TTAACCGGGCTCAGGCCTATGGTGAAGGAAGAAGACAAGG 双分子荧光互补
Bimolecular fluorescence complementationpSPYCE-PtCOL5-R GAGCGGTACCCTCGAGATAAGATGGAACAACTCCATATCCT 
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