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林业科学 ›› 2022, Vol. 58 ›› Issue (6): 56-65.doi: 10.11707/j.1001-7488.20220606

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84K杨PagMSBP1/2a基因对木质素合成的影响

胥雅静,王佳伟,赵岩秋,江成*,黄李超,安轶,曾为,张进,卢孟柱   

  1. 浙江农林大学林业与生物技术学院 省部共建亚热带森林培育国家重点实验室 杭州 311300
  • 收稿日期:2021-03-23 出版日期:2022-06-25 发布日期:2022-09-24
  • 通讯作者: 江成
  • 基金资助:
    浙江省“十四五”育种专项林木协作组课题(2021C02070-1)

Effect of PagMSBP1/2a Gene of 84K Poplar on Lignin Biosynthesis

Yajing Xu,Jiawei Wang,Yanqiu Zhao,Cheng Jiang*,Lichao Huang,Yi An,Wei Zeng,Jin Zhang,Mengzhu Lu   

  1. State Key Laboratory of Subtropical Silviculture School of Forestry and Biotechnology, Zhejiang A & F University Hangzhou 311300
  • Received:2021-03-23 Online:2022-06-25 Published:2022-09-24
  • Contact: Cheng Jiang

摘要:

目的: 膜类固醇结合蛋白(MSBP)是一类定位在细胞质膜上的类固醇受体蛋白,通过响应激素调节信号、参与类固醇代谢影响植物生长发育过程。研究杨树MSBP对生长发育过程的影响,尤其是对木材形成过程的作用,为杨树木材品质改良提供支撑。方法: 以拟南芥MSBP1蛋白序列在毛果杨、银腺杨84K基因组中进行同源序列比对,获取杨树MSBP的核苷酸和氨基酸序列,构建系统进化树并绘制基因结构图谱;利用qPCR分析杨树MSBP基因在84K杨顶芽、根、茎、叶中的相对表达量;在AspWood表达谱数据中分析同源基因在木材形成中的表达模式;构建35 SGFP-PagMSBP1/2a植物表达载体,通过农杆菌介导瞬时转化烟草叶片,激光共聚焦显微镜观察其亚细胞定位情况;构建35 SPagMSBP1/2a植物表达载体,利用农杆菌介导的叶盘法转化84K杨,通过震荡切片观察转基因和对照植株茎段木质部差异;采用乙酰溴法和间苯三酚染色法,分析转基因和对照植株的木质素含量以及木质素在杨树茎段维管组织中的分布。结果: 同源比对鉴定出杨树7个MSBP成员,根据进化关系和基因结构对杨树基因进行命名,其中与AtMSBP1、AtMSBP2同源基因分别命名为PtMSBP1/2aPtMSBP1/2bPtMSBP1/2cPtMSBP1/2aAtMSBP1同源性最高;qPCR结果显示,在根、茎和叶中3个基因均有表达,PagMSBP1/2c的表达量显著低于其他2个基因,PagMSBP1/2a在木质化程度高的老茎(6~8节间)中表达量最高,均为木质化低的幼茎(1~3节间)的6倍,与AspWood数据库所显示的PtMSBP1/2a在木材形成过程中表达量较高且在木质部高表达一致。克隆84K杨的MSBP1/2a(PagMSBP1/2a),其编码的蛋白在N端含有跨膜结构域,亚细胞定位分析表明PagMSBP1/2a定位于内质网膜。构建PagMSBP1/2a超表达载体,通过农杆菌介导的叶盘法转化84K杨树获得转基因植株,转基因植株生长变缓,株高矮于对照;PagMSBP1/2a过表达的植株木质素含量显著低于未转基因对照,乙酰溴可溶性木质素含量降低高达34.6%。结论: 杨树PagMSBP1/2a是定位在内质网膜上的类固醇受体蛋白,参与调控杨树木材形成过程中木质素的生物合成过程,可作为靶基因进行木质素含量调控。

关键词: PagMSBP1/2a基因, 银腺杨84K, 亚细胞定位, 过量表达, 木质素

Abstract:

Objective: Membrane steroid-binding protein (MSBP) is a type of membrane-associated steroid receptor protein located on the cytoplasmic membrane, and it affects multiple processes of plant growth and development by initiating cell response to hormone regulatory signals, regulating steroid transport and metabolism. This study aims to study the role of MSBP during the poplar growth and development, especially its effect on wood formation, so as to provide a basis for the improvement of poplar wood quality. Method: AtMSBP1 protein sequence of Arabidopsis was used for homologous sequence alignment against the genome database of Populus trichocarpa and P. alba × P. glandulosa '84K' to obtain the nucleotide and amino acid sequences of poplar MSBP, and then aphylogenetic tree was constructed and the gene structure was analyzed. qPCR was used to analyze the tissue specific expression of MSBP genes in top buds, roots, stems and leaves of 84K poplar. The expression patterns of P. trichocarpa homologous genes in wood formation tissues were searched against the Aspwood database. The 35 SGFP-PagMSBP1/2a plant expression binary vector was constructed and transiently transformed into tobacco leaves by Agrobacterium. The subcellular localization of PagMSBP1/2a was observed by laser confocal microscope. 35 SPagMSBP1/2a plant expression binary vector was constructed and transformed into poplar 84K by Agrobacterium-mediated leaf disk method. The difference of xylem between PagMSBP1/2a-OE and control plants was investigated through cross-sections examined with microscopy. The content of lignin and the distribution of lignin in the vascular tissues of transgenic and control poplar stems were analyzed by acetyl bromide method and phloroglucinol staining respectively. Result: In this study, 7 poplar MSBP members were identified through homologous comparison, and the poplar genes were named according to evolutionary relationship and gene structure. Ameng them the homologous genes of AtMSBP1 and AtMSBP2 in poplar were named PtMSBP1/2a, PtMSBP1/2b and PtMSBP1/2c respectively, and PtMSBP1/2a had the most homology with AtMSBP1. The result of qPCR analysis showed that the three genes were all expressed in roots, stems, and leaves. The expression of PagMSBP1/2c in tissues was significantly lower than that of PagMSBP1/2a and PagMSBP1/2b, while the expression of PagMSBP1/2a was the highest in old stems, which was nearly 6 times higher than that in young stems. It is consistent with the high expression of PtMSBP1/2a in xylem during the process of wood formation as shown in the Aspwood data. The MSBP1/2a ortholog (PagMSBP1/2a) of 84K poplar was cloned and its encoded protein contained the trans-membrane domain at the N-end. The subcellular localization analysis showed that PagMSBP1/2a was localized in the endoplasmic reticulum membrane. The vector used for over-expression of PagMSBP1/2a was constructed and the gene was transformed into 84K poplar by Agrobacterium mediated leaf disk method. The transgenic poplars more grew slow and were shorter than the control. The lignin content in stems of PagMSBP1/2a overexpressed poplar was significantly lower than that of the control, and the soluble lignin content of acetylbromide decreased by 34.6%. Conclusion: PagMSBP1/2a is a steroid receptor protein located on the endoplasmic reticulum membrane, which is involved in regulating the lignin biosynthesis during the wood formation of poplar. Therefore, it can be used as a target gene in the poplar breeding for lignin content alteration.

Key words: PagMSBP1/2a, Populus alba × P. glandulosa '84K': subcellular location, overexpression, lignin,  

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