毛果杨NAC128基因在次生壁形成中的功能

doi:10.11707/j.1001-7488.20201107

摘要/Abstract

摘要：

目的: 克隆并鉴定与毛果杨次生壁形成相关的NAC（NAM，ATAF1/2，CUC2）转录因子，在组织表达特异性分析的基础上，通过过量表达分析转基因植株表型及下游调控网路，解析该基因调控杨树次生壁组分生物合成的作用机制，为利用该基因进行林木种质创新奠定基础。方法: 基于前期转录组数据，克隆到1个在毛果杨茎中高表达的NAC转录因子PtrNAC128。利用qRT-PCR技术分析PtrNAC128在毛果杨根、茎、叶中的相对表达量。构建35S∷PtrNAC128-GFP植物表达载体，利用农杆菌介导法转化毛白杨；利用基因枪法将PtrNAC128-GFP融合蛋白在烟草叶片中进行瞬时表达，而后对PtrNAC128进行亚细胞定位分析；对毛白杨转基因植株和野生型植株的横切面切片染色观察，并通过qRT-PCR检测木质素、纤维素、半纤维素合成途径关键酶基因及调控次生壁各组分生物合成的NAC、MYB转录因子的表达差异，解析过表达PtrNAC128对杨树次生壁各组分的影响。结果: 毛果杨PtrNAC128与拟南芥ANAC075聚在一支，具有典型NAC结构域和完整的C端激活区。qRT-PCR结果表明PtrNAC128在毛果杨根、茎、叶中均有表达；在老茎中表达最高，是嫩叶中的23.49倍；在嫩茎中也有较高表达，是嫩叶的11.44倍。亚细胞定位分析表明PtrNAC128定位于细胞核内。PtrNAC128过表达毛白杨转基因株系，次生木质部显著增厚，达到野生型的1.42~1.51倍；木质部细胞层数增加，约为野生型的1.22~1.31倍；转基因株系Klason木质素含量较野生型植株增加12%~22%，纤维素含量较野生型植株增加7.4%~13.1%，但半纤维素含量无显著差异；qRT-PCR检测结果表明，PtrNAC128显著提高木质素和纤维素合成途径关键酶基因以及部分次生壁相关NAC、MYB转录因子的表达。结论: 毛果杨PtrNAC128通过激活木质素和纤维素生物合成的关键酶基因及转录因子调控次生壁组分合成。

关键词: 毛果杨, PtrNAC128, 转基因, 次生壁组分

Abstract:

Objective: The aim of this study was to clone and identify NAC (NAM, ATAF1/2 and CUC) transcription factors related to secondary cell wall formation in Populus trichocarpa. Based on the analysis of tissue-specific expression pattern, the phenotype and downstream regulatory network of transgenic plants, our results provide some new insights into the mechanism of the gene regulatory network of secondary cell wall biosynthesis in poplar, laying a foundation for the use of this gene in forest germplasm innovation. Method: Based on the previous transcriptome data, PtrNAC128, which was preferentially expressed in the stems of P. trichocarpa, was cloned. Relative expression of PtrNAC128 in roots, stems and leaves were analyzed by qRT-PCR analysis. The subcellular localization of PtrNAC128 was analyzed by gene gun bombardment method; 35S∷PtrNAC128-GFP plant binary expression vector was constructed and transformed to Populus tomentosa. Anatomical cross-sections of transgenic and wild-type plants were observed by paraffin analysis method, and the effects of PtrNAC128 overexpressing on the components of poplar secondary cell wall were examined. The expression fluctuation of key enzyme genes and NAC, MYB transcription factors which regulating the biosynthesis of lignin, cellulose and hemicellulose synthesis were detected by qRT-PCR analysis. Result: PtrNAC128 clustered in one branch with Arabidopsis ANAC075, with typical domains and intact C terminal activation regions. PtrNAC128 was expressed in all tissues tested, with the highest expression level in old stems and the lowest expression level in leaves. The expression of PtrNAC128 in old and young stems was 23.49 folds and 11.44 folds higher than that in young leaves, respectively. Subcellular localization analysis indicated that PtrNAC128 was localized into the nucleus. The secondary xylem width of PtrNAC128-overexpression (PtrNAC128-OE) transgenic plants had significantly increased to 1.42-1.51 folds, while the number of secondary xylem cells increased to 1.22-1.31 folds compared to the wild-type plants. The Klason lignin contents in the stems of PtrNAC128-OE lines were significantly increased by 12%-22% compared to the wild-type plants. Moreover, no significant effect was detected on the hemicellulose biosynthesis, while cellulose content increased by 7.4%-13.1% in the PtrNAC128-OE lines. The qRT-PCR analysis revealed that the relative expression levels of key synthetic enzyme genes of lignin and cellulose as well as some secondary wall-associated NAC and MYB transcription factors were elevated in the PtrNAC128-OE lines. Conclusion: These results suggested that PtrNAC128 may positively regulate the secondary cell wall biosynthesis by activating the enzyme genes and transcription factors of lignin and cellulose biosynthesis in poplar.

Key words: Populus trichocarpa, PtrNAC128, transgenic, secondary cell wall component

中图分类号:

S718.46

李媛,陈金焕,金曌,侯景丫,姜玉松,邢海涛. 毛果杨NAC128基因在次生壁形成中的功能[J]. 林业科学, 2020, 56(11): 62-72.

Yuan Li,Jinhuan Chen,Zhao Jin,Jingya Hou,Yusong Jiang,Haitao Xing. Functions of NAC128 Gene from Populus trichocarpa in Secondary Cell Wall Formation[J]. Scientia Silvae Sinicae, 2020, 56(11): 62-72.

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基因名称 Gene name	正向引物序列 Forward primer sequence (5′—3′)	反向引物序列 Reverse primer sequence (5′—3′)
克隆和载体构建引物Primers for cloning and vector construction
PtrNAC128	ATGACTACTAAGAGTAATATGGCTTCC	GACCAACCCATGATGATCCTGGTTGTC
PtrNAC128 (BglⅡ, SpeⅠ)	GCGAGATCTGATGACTACTAAGAGTAATATGGCTTCC	GGACTAGTGACCAACCCATGATGATCCTGGTTGTC
定量RT-PCR引物Primers for qRT-PCR
PtrNAC128	GGATAAGAGAGGCTTCAGTAGC	TCAAGAATGATGGAGGTGTG
PtoCCOAOMT1	CAAGAGGTTGATTGAGCTTG	GGTCAGCAGCAAGTGCCTTG
PtoCCR2	CTGTTCAAGCTTATGTGCATG	GTGGAGAACGCTCTCAGAGC
PtoCOMT2	CATGAAGTGGATATGCCATG	GTTGAATGCACAGCACATTAC
PtoC3H3	GAGGTTCCTGGAGGAGGATG	GGAGTCGTCATGTAAGTGAC
PtoPAL4	CCTACATTGACGATCCTTGCAG	GACCTGCATTCCTTGATCCTG
PtoHCT1	ATCAGCATGTAAGGCACGCGG	TGCCAAAGTAACCAGGTGGAAGCGT
PtoCAD1	CAAGCTGATCTTGATGGGTG	CGAATCTATATCTCACATC
Ptr4CL5	CATCCGAGGTGATCAGATCATG	CACAGCAGCATCAGATATCC
PtrF5H2	GAGTCCAGCAAGAGCTCGCAG	GCATAAGCATTGATCATCAC
PtoCesA2B	AGGTTAAGATGGAGCGG	ACGAGGTTGATGATCAAGCC
PtoCesA3A	CGGATATGGATCATGGGGTCC	GGATAGAGATGGACAATGAC
PtoGT8D	GTGCCTGGGCTTATGGCATG	CTAGCCAAGGCTTTGCTCGACC
PtoGT43B	CTCAATCCTCTGGGATCCTG	GTCTCGTCTTCAAGAGCTAC
PtoGT43D	GGTAGAGCCACTTGGGAGC	CACAAGGCAGTATCTCTG
PtoCSE1	GACAGTCCACGGCACGGCTG	CCTCTCAACCCTCTCGTC
PtoMYB192	CGTGACCGAAATCCCATTCGAG	GAGTGGCATGTCATGCTG
PtoMYB161	CGAGCAGAAACCTTCCTTCTC	CCAGCCCTGCTCTTAGGC
PtoNAC156	CCACTCTTGTCGAGTAC	CTTCTCAATGTATCCTGCC
PtoMYB152	GAAGACTTGCTACTGCCAGAT	TCATTCTTGAGCACTGATTG
PtoMYB028	CGTGGTAACTTCAGCAAAGAG	GAGGAGGTTGAGGAGGATTC
PtoMYB090	GAGTTGGTTGAGAAGTATGGC	CGGTGAGAAGCAAGAAGTCT
PtoMYB175	GCTGTGAAGAACCATTGGC	TGCTCATTGACTAACAACGC
PtoNAC105	GGATAACTTGCCCTTCTTGTG	CACCTTTGCTTCTAAATGCC
PtoNAC154	CAAGACCAGGCAAGAATCC	GATGGAAGAAGTGGCGAA
Pto18S	GGCATGGAAGGTGATGCAGATC	CTGTGTCAAACAAGAACTTGTCC

次生壁组分 Secondary cell wall composition	野生型 Wild type	过表达PtrNAC128株系 PtrNAC128 overexpression plants
次生壁组分 Secondary cell wall composition	野生型 Wild type	OE-L3	OE-L6	OE-L7
木质素Lignin
乙酰溴溶解AcBr-soluble/(mg·g^-1)	215.6±4.4	223.9±6.5^*	258.2±6.6^*	238.4±6.9^*
Klason+酸溶Klason+acid-soluble/(mg·g^-1)	217.8±4.4	242.8±10.7	263.7±14.4^*	255.9±8.6^*
Klason/(mg·g^-1)	189.7±2.2	212.7±5.8	231.9±4.1^*	225.4±4.3^*
酸溶Acid-soluble/(mg·g^-1)	28.1±2.2	30.1±4.0	31.8±4.2^*	30.5±4.1
半纤维素Hemicellulose/(mg·g^-1)	155.2±5.1	155.9±8.1	159.6±5.7	152.1±4.2
纤维素Cellulose/(mg·g^-1)	443.6±12.5	476.4±13.5^*	501.5±17.2^*	488.8±16.6^*

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