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PBJ | 北京林业大学康向阳课题组揭示植物DNA单碱基损伤修复新机制

  • 2026-02-02 14:51:53
PBJ | 北京林业大学康向阳课题组揭示植物DNA单碱基损伤修复新机制
近日,Plant Biotechnology Journal 杂志在线发表了由北京林业大学康向阳课题组完成的研究论文“The PagDMG6341–PagWD40–PagPOLD4 Module Coordinates Base Excision Repair in ‘84K’ Poplar (Populus alba×P. glandulosa)。该研究鉴定了 2 个银腺杨‘84K’的 DNA 碱基损伤修复响应基因,即DNA 糖苷酶家族基因 PagDMG6341 和 DNA 聚合酶 δ 家族基因 PagPOLD4;进一步研究证明 WD40 repeat-like 蛋白 PagWD40 是连接 PagDMG6341 与 PagPOLD4 的支架蛋白,从而揭示了PagWD40 作为支架蛋白协同 DNA 糖苷酶和 DNA 聚合酶 δ 亚基参与植物 DNA 单碱基损伤修复的新机制。

DNA 在其不断复制并传递给生物子细胞的过程中难免会受到损伤,主要诱因包括活性氧、代谢中间产物等内源性因素以及紫外线、病原侵染等外源性因素 (Tuteja et al., 2009)。为了生存并将完整的遗传信息忠实地传递给下一代,细胞进化出了一系列DNA损伤修复机制,以应对 DNA 双链断裂、单链断裂以及碱基损伤等DNA 损伤 (Lucchesi, 2018)。其中,碱基损伤是最为高频发生的损伤类型之一,其最主要的修复途径为碱基切除修复 (Base Excision Repair, BER) (Sidhu, 2023)。BER 修复的过程主要包括特异性 DNA 糖苷酶识别并切除损伤碱基,招募相关修复蛋白完成缺口的填补以及后续的封口 (Grin et al., 2023)。碱基损伤依据受损碱基的数量可分为 long-patch (LP-BER) 和 short-patch (SP-BER) 两类。其中,单功能型 DNA 糖苷酶主导 long-patch (LP-BER),双功能型 DNA 糖苷酶则主导 short-patch (SP-BER) (Roldán-Arjona et al., 2019)。在哺乳动物中,这些修复环节需要 Pol β、Pol δ、Pol ε 等 DNA 聚合酶和 XRCC1 或 PCNA 这类支架蛋白协调完成 (Kubota et al., 1996)。近年来的研究表明植物中同样具备完整的 SP- 和 LP-BER 修复机制。研究表明,AthPol λ 与 SP-BER 有关,是 Pol β 的替代因子 (Morales-Ruiz et al., 2024)。Pol δ (Wang et al., 2019) 和 Pol ε (Pedroza-García et al., 2017) 则可能在 LP-BER 中发挥作用。然而,对于高等植物中 DNA 碱基损伤的修复组件的招募与互作机制尚存在未知。
北京林业大学康向阳教授团队通过研究证明了 PagDMG6341-PagWD40-PagPOLD4 模块在银腺杨‘84K’ DNA 碱基损伤修复中的重要作用,揭示了植物 BER 通路中 PagWD40 蛋白作为 DNA 糖苷酶和聚合酶 δ 亚基的支架蛋白的新机制,为植物 DNA 损伤修复网络的构建提供了新思路。全文主要研究结果如下:

1.      5-AU 诱导银腺杨‘84K’ DNA 损伤及其相关基因表达动态

对银腺杨‘84K’分化培养叶片施加5-氨基尿嘧啶 (5-AU) 同步化处理后,每隔 12h取样,其中,5-AU 处理时期包括 2 个取样点(Ta 和 Tb),脱药时期包括 4 个取样点(Ha、Hb、Hc 和 Hd),每个取样点的样本包括处理组和对照组,每组3个生物学重复,总计 36 个样本(图1.A)。利用流式细胞术对叶片伤口处的分化细胞周期进行测定并统计细胞周期的 G1, S 以及 G2 期细胞占比(图1.B, C 和D),在处理时期以及脱药时期观察到了 S 期(图1.C)和 G2/M 期(图1.D)细胞富集现象。

主成分分析 (PCA) 结果表明,每个样品的生物学重复之间具有较高的相似性,不同处理的叶片具有不同的基因表达模式,适合进一步分析(图1. E)。分层聚类结果表明,除了 5-AU 处理前期(Ta VS CKa)以及脱药后期(Hd VS CKf)基因表达的模式在处理和对照组之间相近,其他时间节点可以明显的依据处理和对照组划分为两个类群(图1.G)。差异表达基因(DEGs)数量随着脱药时长呈明显的下降趋势(图1.F)。通过Fuzzy聚类分析,将所有样本的差异表达基因划分了 8 个模式(C1-C8),其中,C2, C4, C6 和 C7 整体呈下降趋势,C1, C3, C5 和 C8 整体呈上升趋势。其中 C2 和 C7 在 5-AU 处理和脱药时期的交替处有显著的波动,C1 和 C8 主要在脱药时期有显著的波动(图1.H)。通过 GO 功能富集分析可以发现 C2 和 DNA 的损伤修复有关,主要涉及单碱基修复、错配修复;C7 主要和细胞壁的改变有关;C1 中的基因主要和 DNA 复制以及微管蛋白有关;C8 中既包括 DNA 修复相关基因也包括和细胞壁合成以及微管蛋白相关的功能基因(图1.I)。

图1. 5-AU 处理期间各样本细胞周期及相关基因表达动态

2.      5-AU 处理过程中相关基因共表达网络分析和关键响应基因鉴定

进一步基于银腺杨‘84K’叶片的转录组数据进行WGCNA分析。共识别出 10 个共表达模块。与细胞周期(G1、S、G2 期)的细胞占比表型进行相关性分析(图2.A, B)结果显示,brown 和 turquoise 两个模块在三种细胞周期表型中均表现出显著相关性(p < 0.05)(图2.B)。对 brown 和 turquoise 模块中差异表达且与表型高度相关的基因进行GO 富集与表达趋势分析(图2.C, D)结果显示,brown 模块的基因在 5-AU 处理时期表达上调,随后逐渐下调,主要涉及 DNA 修复、防御反应、转录调控以及质膜组分等功能(图2.C);而 turquoise 模块基因则在脱药时期显著上调,主要富集了大量细胞周期相关因子,以及与 DNA 修复、防御、转录及细胞结构重塑相关的基因(图2.D)

构建共表达网络图(图2.E)分析结果表明,在 brown 模块中与 DNA 修复相关且具有较高的加权度的基因有 4 个,其中 3 个为 AT5G44680 的剪切变体;另一个为 TTN7,属于染色体结构维持蛋白家族,对于生物体姐妹染色单体的排列至关重要。在 turquoise 模块中,共鉴定出  2 个与DNA 修复相关基因, 分别为 POLD4 和 EXO1B,二者均与多个细胞周期调控基因以及防御响应基因相关,其中 POLD4 具有较高的加权度。

结合 KEGG 功能注释结果,重点聚焦于 AT5G44680 和 POLD4 两个关键基因。其中 AT5G44680  在 KEGG 数据库中被注释为原核生物碱基切除修复通路 (BER) 中的 Tag 基因同源物,具有 DNA-3-甲基腺嘌呤糖基酶 I 活性,其对 DNA 单碱基错配有识别和切除的作用;POLD4  是 BER 的修复复合物成员,具有修复 AP 缺口的作用 (Roldán-Arjona et al., 2019)(图2.F),二者在 BER 途径中有潜在的协同作用关系。随后通过 RT-Qpcr 确定了 AT5G44680 和 POLD4 先后差异高表达(图2.G, H)

图2. DNA 损伤修复过程中银腺杨‘84K’叶片细胞相关基因的共表达网络分析

3.      PagDMG6341 和 PagPOLD4 的细胞型特异性表达、亚细胞定位及蛋白序列分析

因 AT5G44680 属于 DNA-3-methyladenine glycosylase I,且在银腺杨‘84K’参考基因组中的基因 ID 为 Pag_A_006341 (Li, 2020), 故将其命名为 PagDMG6341。通过分析处于 S  期高峰期的离体分化培养 3 天的叶片单细胞测序数据,利用 UMP 降维可将叶片组织分成了 8 个细胞簇,分别属于表皮细胞、保卫细胞、叶肉细胞、增殖细胞以及维管细胞,发现 PagDMG6341 和 PagPOLD4 都主要在增殖细胞簇特异性表达(图3.A-C)。在烟草中瞬时转化 35S::PagDMG6341-GFP,35S::PagPOLD4-GFP 和 35S::GFP 三个表达载体(图3.D),结果表明 35S::PagDMG6341-GFP 和 35S::PagPOLD4-GFP 两个融合蛋白和 35S::GFP 对照蛋白均可定位于细胞核和细胞膜(图3.E)

将 PagDMG6341 和 PagPOLD4 蛋白的序列与其同家族以及不同物种中的同源蛋白序列进行比较,发现这两个蛋白序列在不同物种中都具有明显的保守性 (图3.F,G),推测其可能具有类似的功能。PagDMG6341 同已有相关报道一样聚类到单功能型糖苷酶 (图3.F)PagPOLD4 聚类到 Pol δ 家族。

图3. PagDMG6341和 PagPOLD4 在银腺杨‘84K’中的细胞型特异性表达、亚细胞定位及蛋白序列分析

4.      沉默表达 PagDMG6341 和 PagPOLD4 显著影响杨树生长发育

为了验证 PagDMG6341 和 PagPOLD4 在杨树 DNA 损伤反应中的作用,用银腺杨‘84K’构建了每个基因的 RNA 干扰系。结果表明,野生型和 RNA 干扰系在根形态和整株植物大小方面存在显著差异(图 4A)PagPOLD4-RNAi 植株偶尔会在茎节或腋芽处长出异位的不定根(图 4B)。对移栽苗施加 5-AU(1.5 mM)叶面喷施,结果表明,大多数 PagDMG6341 和 PagPOLD4-RNAi 系列植株对 5-AU 应激高度敏感。与野生型相比,PagDMG6341-RNAi(HR = 13.13,P < 0.05)和 PagPOLD4-RNAi 植株(HR = 16.53,P < 0.0001)的死亡率显著增加(图 4C)。在少数存活的转基因植株(例如 PagDMG6341-RNAi-3、-6、-8 和 PagPOLD4-RNAi-11)中,其生长也明显迟缓(图 4D、E)。RT-qPCR 分析显示,与野生型相比,两种 RNAi 系列植株中细胞周期基因 CYCA2;1 和 CYCB2;1 以及细胞壁重塑基因 XTH32 和 XTH33 的转录水平显著下调(图 4F-11),表明细胞周期进程受损以及细胞壁重塑能力降低。相比之下,与生长素调控相关的基因 ARF19 和 NPH4 的表达水平并无显著差异(图 4J、K)

图4. 银腺杨‘84K’PagDMG6341-RNAi 和 PagPOLD4-RNAi 株系表型及 5-AU 敏感性

5.      PagDMG6341 和 PagPOLD4 在蛋白水平上与 PagWD40互作

分别使用 PagDMG6341 和 PagPOLD4 作为诱饵蛋白,在酵母双杂交(Y2H)文库中进行筛选实验,并将两个诱饵蛋白筛库结果的交集作为候选的猎物蛋白。随后使用 AlphaFold 3 对候选蛋白进行预测,筛选出一个显著与两个诱饵蛋白潜在互作的 WD40 repeat-like 蛋白——Pag_G_011820(此后命名为 PagWD40),其在互作界面可预测到多个氢键,表明具有物理结合的可能性(图 5A)。通过 Y2H 点对点实验、双分子荧光互补(BiFC)和分裂荧光素酶互补(LCI)等方法进行验证,结果证实 PagWD40 与 PagDMG6341 和 PagPOLD4 存在相互作用(图 5B、C 和 D)。值得注意的是,PagWD40 与 PagDMG6341 的相互作用在细胞核和质膜中均有观察到,而与 PagPOLD4 的相互作用主要局限于细胞核(图 5C)。进一步通过酵母三杂交(Y3H)实验证明,在 SD/-Leu-Trp-His + 3-AT(80 mM)条件下(该条件抑制 PagWD40 的表达),酵母无法生长;而在 SD/-Leu-Trp-His-Met + 3-AT(80 mM)的条件下(该条件可诱导 PagWD40 的表达),酵母生长正常,表明 PagWD40 的存在是介导 PagDMG6341 与 PagPOLD4 之间联系所必需的(图 5E)这些发现表明,PagWD40 作为 PagDMG6341 和 PagPOLD4 之间的分子“桥梁”,在细胞 DNA 碱基切除修复过程中协同发挥作用。

图5. PagWD40 在蛋白水平上与 PagDMG6341 和 PagPOLD4 相互作用

该研究发现了两个在植物 BER 途径中起关键作用的基因,即 PagDMG6341 和 PagPOLD4,它们在碱基修复过程的不同阶段发挥着作用。PagWD40 作为一种支架蛋白,在 LP-BER 中形成 PagDMG6341-PagWD40-PagPOLD4 的功能复合物,并发挥 DNA 单碱基损伤修复作用。该工作揭示了一种新的植物 DNA 碱基修复机制,并扩展了 WD40 repeat-like 蛋白在基因组维护中的功能范围。
北京林业大学博士研究生凌傲宇与金亦佳为论文共同第一作者,北京林业大学康向阳教授(https://biology.bjfu.edu.cn/szdw/lmycyz/js2/848489073ec94a7aa2ede99b344a0bb1.htm)、张平冬教授(https://biology.bjfu.edu.cn/szdw/lmycyz/js2/09087fccc0db4df98c733f8160e691ef.htm)为该研究工作共同通讯作者。北京林业大学杜康讲师、夏宇飞博士后、以及博士研究生江慎秀、舒江海、胡晓桐等也为该项研究做出重要贡献。研究工作得到了“十四五”国家重点研发计划课题“林木四倍体高效诱导技术研究”(2021YFD2200104)的支持。
论文链接:https://onlinelibrary.wiley.com/doi/10.1111/pbi.70543

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  1. CONNECT:[ UseTime:0.000434s ] mysql:host=127.0.0.1;port=3306;dbname=h_mffb;charset=utf8mb4
  2. SHOW FULL COLUMNS FROM `fenlei` [ RunTime:0.000814s ]
  3. SELECT * FROM `fenlei` WHERE `fid` = 0 [ RunTime:0.000316s ]
  4. SELECT * FROM `fenlei` WHERE `fid` = 63 [ RunTime:0.000266s ]
  5. SHOW FULL COLUMNS FROM `set` [ RunTime:0.000494s ]
  6. SELECT * FROM `set` [ RunTime:0.000175s ]
  7. SHOW FULL COLUMNS FROM `article` [ RunTime:0.000622s ]
  8. SELECT * FROM `article` WHERE `id` = 462222 LIMIT 1 [ RunTime:0.000708s ]
  9. UPDATE `article` SET `lasttime` = 1770940994 WHERE `id` = 462222 [ RunTime:0.001927s ]
  10. SELECT * FROM `fenlei` WHERE `id` = 66 LIMIT 1 [ RunTime:0.000292s ]
  11. SELECT * FROM `article` WHERE `id` < 462222 ORDER BY `id` DESC LIMIT 1 [ RunTime:0.000428s ]
  12. SELECT * FROM `article` WHERE `id` > 462222 ORDER BY `id` ASC LIMIT 1 [ RunTime:0.003601s ]
  13. SELECT * FROM `article` WHERE `id` < 462222 ORDER BY `id` DESC LIMIT 10 [ RunTime:0.000745s ]
  14. SELECT * FROM `article` WHERE `id` < 462222 ORDER BY `id` DESC LIMIT 10,10 [ RunTime:0.001386s ]
  15. SELECT * FROM `article` WHERE `id` < 462222 ORDER BY `id` DESC LIMIT 20,10 [ RunTime:0.001330s ]
0.078373s