Identification and Expression Analysis of <i>NF-Y</i> Gene Family in <i>Pseudostellaria heterophylla</i>

LI Jun; ZHOU Tao; WANG Chong-min; LIU Xiao-qing

doi:10.13718/j.cnki.xdzk.2021.08.004

2021 Volume 43 Issue 8

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LI Jun, ZHOU Tao, WANG Chong-min, et al. Identification and Expression Analysis of NF-Y Gene Family in Pseudostellaria heterophylla[J]. Journal of Southwest University Natural Science Edition, 2021, 43(8): 28-35. doi: 10.13718/j.cnki.xdzk.2021.08.004

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LI Jun, ZHOU Tao, WANG Chong-min, et al. Identification and Expression Analysis of NF-Y Gene Family in Pseudostellaria heterophylla[J]. Journal of Southwest University Natural Science Edition, 2021, 43(8): 28-35. doi: 10.13718/j.cnki.xdzk.2021.08.004

Identification and Expression Analysis of NF-Y Gene Family in Pseudostellaria heterophylla

Resource Institute for Chinese & Ethmic Materia Medica, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China

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Received Date: 06/06/2020
Available Online: 20/08/2021
MSC: S567; R282

Abstract

Nuclear factor Y (NF-Y) is an important transcription factor in eukaryotes. In this study, bioinformatics was used to identify NF-Y protein family members from the transcriptome database of P. heterophylla, and the transcriptome data were used to analyze the expression level of each gene in different tissues. Their response to soil water stress and sucrose was also studied. Three subfamilies of NF-Y protein family of P. heterophylla were identified by searching three transcriptome databases, including 9 members of PhNF-YA, 10 members of PhNF-YB and 5 members of PhNF-YC. It was found that 9 members, such as PhNF-YA4, were highly expressed in leaves. High expression level of PhNF-YA3 and PhNF-YB3, PhNF-YB8 were observed in bark and xylem of tuberous root, respectively. The expression of 11 members in the tuberous root of P. heterophylla was significantly up-regulated under drought stress. Two members had up-regulated expression in microtuberous root along with the increase of soil water content. Most genes could respond to content change of sucrose. These results suggest that some factors can affect the expression level of NF-Y protein in organs, change the interaction between different NF-Y proteins in plants, and regulate the morphogenesis of organs and response to stresses.
- Pseudostellaria heterophylla,
- NF-Y protein,
- transcriptome,
- expression analysis

References

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沈阳化工大学材料科学与工程学院沈阳 110142

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核因子Y(nuclear factor Y，NF-Y)，又称CBF或亚铁血红素激活蛋白HAP，主要由NF-YA，NF-YB及NF-YC 3个亚家族组成^[1]. NF-Y蛋白家族的不同成员可通过不同途径进入细胞核，其中NF-YA是由Importin β介导输入核中，而含有HFM亚基的NF-YB和NF-YC作为异二聚体复合物则由Importin 13介导进入细胞核^[2]. 3个亚家族可以单独调控基因表达，也可形成NF-YB/NF-YC CONSTANS或NF-Y复合体，进一步结合CCAAT元件参与基因表达的调控^[3].

在哺乳动物及酵母体内，NF-Y的3个亚基均由单基因编码^[4]，但植物中每个亚基均由基因家族编码^[5]，提供了更多的NF-YA/NF-YB/NF-YC三聚体组合，增加了核因子Y功能的复杂性，参与了在种子萌发、胚胎发育、开花过程以及植物对氮、磷、脱落酸(ABA)以及盐分胁迫等的响应过程^[6]. 研究表明大豆GmNF-YCa在蔗糖和甘露醇诱导后上调表达，过表达可显著改善甘露醇和蔗糖胁迫下拟南芥种子的发芽率、根长和侧根伸长明显等^[7]. 谷子SiNF-YA6主要在根部表达，同时受低氮、干旱、高盐和ABA等胁迫的诱导表达，该基因的过表达显著提高了拟南芥对低氮胁迫的耐受性^[8]. 由此可见，NF-Y蛋白家族的不同成员不仅参与了植物多种生长发育进程，且在植物抵御逆境胁迫等方面具有重要作用.

太子参为传统中药，系石竹科植物孩儿参Pseudostellaria heterophylla (Miq.) Paxex Pax et Hoffm的干燥块根，含有多糖类、皂苷类、磷脂类、环肽类等活性成分，具有增强免疫力、降血糖、抗疲劳等多种功效^[9]. 随着近年来太子参需求量的逐年上升，逐步转向人工种植，主要分布于温暖湿润的气候环境中^[10]. 高温、干旱等逆境容易引起太子参生长发育受阻，从而导致减产、病害等问题. 因此，研究NF-Y基因家族的表达模式及对干旱胁迫的响应，对于深入理解其在太子参生长发育过程中的功能和作用，解析NF-Y蛋白在植物抵抗逆境胁迫中的作用机制具有重要意义.

1. 材料

供试材料为贵州施秉常用种，转录组数据由上海美吉生物医药科技有限公司测序完成. 数据库1：采用课题组前期构建的太子参转录组数据库(叶、茎、根表皮、根木质部)^[11]；数据库2：参照路丙社等^[12]的方法，设置30%，44%，58%，72%，86%及100%的土壤水分梯度，相对含水量=土壤含水量/田间持水量×100%，取块根进行转录组测序，de Novo组装；数据库3：采用0.2 mg/L NAA(萘乙酸)诱导组培苗生根^[13]，然后转入MS+45 g/L蔗糖，MS+60 g/L蔗糖及MS+75 g/L蔗糖的培养基中培养，取块根进行转录组测序，de Novo组装.

2. 方法

2.1. NF-Y基因家族的鉴定

从NCBI中下载甜菜(Beta vulgaris L.)、拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa L.)等的NF-Y蛋白家族成员. 通过BlastP分别比对3个数据库，从中鉴定太子参NF-Y家族同源序列，期望值设为小于0.001，结合数据库原有的注释结果，剔除注释错误的序列，将3个数据库中鉴定的NF-Y蛋白序列统一编号.

2.2. 系统进化树的构建及蛋白保守序列比对

采用muscle 3.8软件(http://www.drive5.com/muscle/)对太子参及甜菜、拟南芥及水稻进行多序列比对，分析结果用MEGA X(https://www.megasoftware.net/)分析，采用邻接法(neighbor-joining method)构建NF-Y的系统进化树，检验参数Booistrap重复1 000次. 利用MEME在线程序(http://meme-suite.org/)对NF-Y蛋白家族的保守基序进行分析，预测太子参NF-Y蛋白家族的蛋白质保守基序(Motif)，参数设置为默认值，并用WebLogo软件(http://weblogo.berkeley.edu/logo.cgi)展示保守基序.

2.3. 表达分析

采用RSEM软件(http://deweylab.biostat.wisc.edu/rsem/)进行基因表达分析，采用RPKM值(reads per kb permillion reads)表示基因的相对表达水平，采用Cluster 3.0软件对NF-Y基因的表达水平进行标准化，最后采用Heatmap图展示基因的表达.

2.4. 蛋白互作分析

采用美吉生物云平台(https://cloud.majorbio.com/)中的STRING数据库(http://string-db.org/)对NF-Y蛋白互作进行分析. 参考拟南芥的蛋白互作关系，构建太子参NF-Y蛋白的互作关系网络. 采用confidence score值评价互作关系的可信度，默认值0.4，值越大代表互作关系越可靠.

4. 讨论

已有证据表明NF-Y蛋白在叶绿体形成及光合作用中具有重要作用. 拟南芥AtNF-YA5，AtNF-YB9和AtNF-YC9形成的复合体可与GCR1，GPA1和Pirin1组成信号传递链，参与调控植株中叶绿素a/b结合蛋白基因的表达^[14]. 过量表达小麦TaNF-YB3后显著提高叶绿素含量、光合速率及早期生长率^[15]. 水稻Os-NF-YB2/3/4可影响多个核酮糖-1，5-二磷酸羧化酶/氧合酶小亚基和叶绿素a/b结合蛋白基因的表达^[16]. 本研究发现太子参PhNF-YA4，PhNF-YA5等6个基因，PhNF-YB8，PhNF-YB10及PhNF-YC2在叶中高量表达，且可能具有复杂的相互作用关系，暗示这些基因在协同调控叶的生长发育及光合作用中具有重要作用，但具体机制尚不明确. 拟南芥NF-YA1/5/6/9共同调控雄配子发育、胚胎发育、种子形态及萌发等^[17-18]，单个基因或两个基因的突变并不能发生表型的改变，但过表达其中任一基因均能影响种子的发育，暗示了太子参PhNF-YA亚家族也有功能冗余的可能性. 此外，PhNF-YA1及PhNF-YB3，PhNF-YB8在块根木质部中有较高表达，且进化分析结果显示PhNF-YA1，PhNF-YA4，PhNF-YA5，PhNF-YA8与AtNF-YA1，AtNF-YA9聚为一类，暗示了这些太子参NF-Y基因可能与太子参块根的发育有一定关系.

NF-Y蛋白在水分胁迫过程中具有重要作用. 研究表明拟南芥大部分NF-YA基因呈上调表达，而小麦中NF-YA则相反^[19-20]. 过表达AtNF-YB1和ZmNF-YB2可显著减少转基因植株的叶片水分流失及提高抗旱能力^[21]. 部分NF-Y基因的转录受ABA途径调控，转录后受到miR169的调控^[22]. 本研究中太子参PhNF-YA1，PhNF-YA4，PhNF-YA5，PhNF-YA7，PhNF-YA9，PhNF-YB2，PhNF-YB3，PhNF-YB8及PhNF-YC1，PhNF-YC3，PhNF-YC4在干旱胁迫下均显著上调，且进化分析结果显示PhNF-YB2，PhNF-YB3，PhNF-YB10与AtNF-YB1，AtNF-YB2等6个拟南芥NF-YB蛋白归入一类，暗示了这些太子参NF-YB蛋白与抗旱有密切联系. 此外，蛋白间互作关系暗示PhNF-YC3，PhNF-YC4在干旱胁迫响应过程中可能具有核心调控作用. 值得注意的是，PhNF-YA6及PhNF-YC2的表达随土壤水分的增加而增加，揭示了这些基因在太子参感知土壤水分变化的过程中具有重要作用.

蔗糖及其衍生物不仅是光合同化物与能量的运输和贮藏形式，也是植物中一种重要的信号分子，调控基因表达及影响器官的生长发育^[23-24]. 本研究分析了太子参NF-Y蛋白对蔗糖胁迫的响应，发现了大部分基因能够响应蔗糖浓度的变化，暗示了环境因子可通过影响组织中蔗糖的分布来调控NF-Y蛋白的表达，进而影响器官的形态建成.

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Identification and Expression Analysis of NF-Y Gene Family in Pseudostellaria heterophylla