紫花苜蓿MsNST的克隆及对木质素与纤维素合成的功能分析

doi:10.3864/j.issn.0578-1752.2020.18.016

摘要/Abstract

摘要：

【目的】木质素和纤维素是植物次生细胞壁的主要组成成分,是影响牧草消化率和品质的主要因素之一。紫花苜蓿作为高蛋白饲草,是奶牛等草食家畜优质饲草的主要来源。因此,苜蓿木质素和纤维素合成机制一直是受到关注的研究热点。模式植物拟南芥NAC家族的NST转录因子（NAC secondary wall thicking promoting factor）调控次生细胞壁的合成,但紫花苜蓿NST的功能与调控机制尚不明确。本研究通过分析紫花苜蓿NST的表达模式,及在拟南芥与苜蓿中过表达揭示其对木质素和纤维素合成的影响。【方法】通过同源克隆获得MsNST CDs序列,并对该基因进行生物信息学分析。利用qRT-PCR检测该基因受赤霉素（GA3）,水杨酸（SA）和多效唑（PCB）诱导后的表达模式。通过转基因植株中过表达MsNST,研究其对木质素和纤维素含量及其合成相关基因的影响。【结果】克隆MsNST 的CDs序列,最大开放阅读框为945bp,编码314个氨基酸。生物信息学分析表明,MsNST主要由无规则卷曲组成（60.83 %）;三级结构预测显示,MsNST以同源二聚体的形式有效的促进蛋白质间的相互作用。进化树分析表明,单子叶和双子叶分为两个分枝,暗示存在一定程度的进化,而MsNST与蒺藜苜蓿和大豆的NST属于双子叶植物分枝中的亚分枝,表明豆科植物间亲缘关系较近。MsNST与拟南芥NST1-3的氨基酸序列相似性较高（49%—55.9%）,并含有NAC转录因子的5个保守结构域。qRT-PCR分析表明,MsNST受GA3、SA和PCB的诱导表达,相对表达水平（12h）分别是对照的2.19、3.67和3.65倍。过表达MsNST导致拟南芥下胚轴缩短,转基因拟南芥半矮化,花序茎束间细胞壁纤维增厚,细胞壁结晶纤维素（13%）、总糖（7%）和木质素（11.7%）含量增加。通过分析木质素和纤维素合成相关基因表达水平发现,拟南芥和苜蓿中过表达MsNST均能激活木质素合成关键基因（PAL,4CL等）及纤维素合酶复合体亚基CesA家族基因的表达。【结论】MsNST受外源激素GA3、SA和PCB诱导表达。转基因植物中过表达MsNST可激活次生壁木质素和纤维素合成相关基因的表达,同时茎束间纤维细胞壁增厚,细胞壁结晶纤维素、总糖和木质素含量增加,暗示MsNST对次生细胞壁木质素和纤维素的合成有重要的调节作用。

关键词: 紫花苜蓿, NST转录因子, 次生壁, 纤维素, 木质素

Abstract:

【Objective】Lignin and cellulose, one of the main components in the secondary cell wall of higher planta, are one of the important factors affecting the quality and digestibility of legume forage alfalfa. The model plant, Arabidopsis thaliana, NST genes belong to the NAC transcription factor family were shown to have a key role in regulating secondary cell wall biosynthesis. However, the function of MsNST in alfalfa was still elusive. In this study, the aim was to investigate the expression patterns of MsNST gene induced by exogenous hormones and its function in lignin and cellulose biosynthesis pathway. 【Method】MsNST was isolated from alfalfa by homology-based cloning. Bioinformatic analysis was conducted by online tools. In order to investigate the functions of MsNST in cell wall cellulose and lignin synthesis pathway, overexpression of MsNST in transgenic alfalfa plants was programed. In this study, qRT-PCR was applied to reveal the expression pattern of genes related to lignin and cellulose biosynthesis pathway, and expression level under gibberellin (GA3), salicylic acid (SA) and paclobutrazol (PCB) treatments. 【Result】 An open reading frame (ORF) of 945 bp encoding MsNST with 314 amino acids was cloned. The similarity analysis of amino acid showed both MsNST and AtNST1-3 shared high sequence homology (from 49% to 55.9%) containing five typical NAC conservation subdomains. Bioinformatic analysis indicated that MsNST was mainly constituted by the random coil (60.83%), and a comparative protein modeling implied that homodimer might promote protein-protein interaction. Phylogenetic analysis revealed that MsNST widely expressed in higher plant species from monocot to dicot. The increased MsNST expression levels were detected with GA3, SA, and PCB (2.19- 3.67 times at 12h) treatment. Overexpression of MsNST in Arabidopsis resulted in shortening of hypocotyl axis, semi-dwarf and interfascicular fiber cell wall thickening and the accumulation of lignin (11.7%), cellulose (13%) and total sugar (7%) compared with wide type. Otherwise, the expression level of other genes in lignin and cellulose biosynthesis pathway, including PAL1, 4CL1, and CesAs were detected by qRT-PCR and the results determined these genes were up-regulated in transgenic plants compared with the control. 【Conclusion】MsNST was inducible by GA3, SA, and PCB, and the overexpression of MsNST in transgenic plants triggered the expression of key genes related to secondary cell wall synthesis, and led to shortened hypocotyl, semi-dwarf and thickened interfascicular fiber cell wall. Additionally, overexpression MsNST accumulated the content of lignin, cellulose and total sugar. These findings suggested that MsNST might play a crucial role in cell wall lignin and cellulose synthesis pathway.

Key words: alfalfa, transcription factor, secondary wall, cellulose, lignin

蒋旭,崔会婷,王珍,张铁军,龙瑞才,杨青川,康俊梅. 紫花苜蓿MsNST的克隆及对木质素与纤维素合成的功能分析[J]. 中国农业科学, 2020, 53(18): 3818-3832.

JIANG Xu,CUI HuiTing,WANG Zhen,ZHANG TieJun,LONG RuiCai,YANG QingChuan,KANG JunMei. Cloning and Function Analysis of MsNST in Lignin and Cellulose Biosynthesis Pathway from Alfalfa[J]. Scientia Agricultura Sinica, 2020, 53(18): 3818-3832.

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引物 Primer	核酸序列（5′-3′） Nucleotide sequence (5′-3′)	功能 Function	文献Reference
MsNST-f	TCAACTTTTTGGGTCCCTTGTG	MsNST克隆 Cloning of MsNST
MsNST-r	TCACCACATGCTATCACCATTG	MsNST克隆 Cloning of MsNST
actin-2s	CAAAAGATGGCAGATGCTGAGGAT	内参基因 Reference gene Actin
actin-2a	CATGACACCAGTATGACGAGGTCG	内参基因 Reference gene Actin
qNST-f	TCATCTCAAAACCCTAGACAGCCC	实时定量PCR qRT-PCR
qNST-r	GTAATTTGCTTCATAATTCTCTTCCTTG	实时定量PCR qRT-PCR
Ntest-f	GCACAATCCCACTATCCTTCG	转基因拟南芥阳性鉴定Identification of transgenic lines
Ntest-r	AGTTTTTTGATTTCACGGGTTGGGG	转基因拟南芥阳性鉴定Identification of transgenic lines
W-NST-F	CATTTGGAGAGAACACGGGGGACTCTAGAATGCCTGATAACATGAGTATAT	Pbi121-MsNST超表达载体构建 Construction of Pbi121-MsNST overexpression vector
W-NST-R	AACATAAGGGACTGACCACCCGGGGATCCTCACCACATGCTTATCAC CATT
Atact-f	GCAACATACGACGAAATCAAGAA	qRT-PCR内参基因 Actin gene for qRT-PCR	[18] [19] [20] [21]
Atact-r	CGACACGAGAACTGTAACCCC	qRT-PCR内参基因 Actin gene for qRT-PCR
AtPAL1-f	ATGGAGATTAACGGGGCACAC	木质素与纤维素合成相关基因的表达分析 Expression analysis of lignin and cellulose biosynthesis related to genes
AtPAL1-r	GTACCGCCGAGAACACCGCC
At4CL1-f	GATTTGAGCTCGATAAGAGTGGTG
At4CL1-r	ATTTGCTAGTTTTGCCCTCA
AtCESA4-f	ATTCTGGGTGATTGGCGG
AtCESA4-r	AATAATGAGAGTTGTCGGAGGG
AtCESA7-f	TTCTTGCCTACTGTATCCTTCC
AtCESA7-r	GCTAACTCCGCTCCATCTCA
AtCESA8-f	CATCCCAACGCTATCAAACCTA
AtCESA8-r	CTGAGACACCTCCAATAACCCA
MsCESA3-f	TCGATGGGCTTTAGGTTCAG
MsCESA3-r	TGAGAAGAGGAATGGAAGTG
MsCESA6-f	CCCTCTTCATATCCATCGCAG
MsCESA6-r	CACCTCCAATCACCCAAAAC
MsCESA7-f	GATGAAGCAAGACAACCACTG
MsCESA7-r	CTGGGTTCATAAGTCTGTATCGG
MsPAL-f	ATGAGGTGAAGCGTATGGTG
MsPAL-r	CATCCCTAGCAGATTCAGACAG
Ms4CL-f	TTCACGTCCTTGCCTCATCA
Ms4CL-r	CCAAGTTTGTTGAGACCGGAGG
MsCOMT-f	AAAGTGATTGTGGCAGAATGCA
MsCOMT-r	TTTTGTGGCCAGGCTTGAA