Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (18): 3818-3832.doi: 10.3864/j.issn.0578-1752.2020.18.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Cloning and Function Analysis of MsNST in Lignin and Cellulose Biosynthesis Pathway from Alfalfa

JIANG Xu(),CUI HuiTing,WANG Zhen,ZHANG TieJun,LONG RuiCai,YANG QingChuan,KANG JunMei()   

  1. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2019-10-08 Accepted:2019-12-26 Online:2020-09-16 Published:2020-09-25
  • Contact: JunMei KANG E-mail:jiangxu2009@yeah.net;kangjunmei@caas.cn

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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

Fig. 1

Cellulose and lignin biosynthesis pathway A．Schematic diagram of cellulose synthesis; B. Schematic diagram of lignin monomer synthesis. CesA, Cellulose Synthase. PAL: phenylalanine ammonia-lyase, C4H: Cinnamate-4-hydrolylase. 4CL: 4-coumarate coenzyme A ligase, COMT: caffeic acid 3-O-methyltransferase. CCR: cinnamyl coenzyme A reductase"

Table 1

Primer sequences used in this study"

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
actin-2s CAAAAGATGGCAGATGCTGAGGAT 内参基因
Reference gene Actin
actin-2a CATGACACCAGTATGACGAGGTCG
qNST-f TCATCTCAAAACCCTAGACAGCCC 实时定量PCR
qRT-PCR
qNST-r GTAATTTGCTTCATAATTCTCTTCCTTG
Ntest-f GCACAATCCCACTATCCTTCG 转基因拟南芥阳性鉴定Identification of transgenic lines
Ntest-r AGTTTTTTGATTTCACGGGTTGGGG
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
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

Fig. 2

Cloning of MsNST from alfalfa A: PCR amplification of MsNST; B: Nucleic acid and deduced amino acid sequences of MsNST"

Fig. 3

Bioinformatics analysis of MsNST A: Hydrophilicity of MsNST protein predicted by Protscale; B: Non-transmamrane domain for MsNST protein predicted by TMHMM; C: Secondary structure of MsNST predicted by SOPMA; D: Prediction and construction of MsNST tertiary structure by SWISS-MODEL; E: Prediction of MsNST subcellular localization by Cell-Ploc"

Fig. 4

Amino acid alignment of MsNST and AtNST1, 2, and 3 Homology level was highlighted by shading in color: black for 100%, grey for ≥50% identity. Conservative domain of NAC was underlined in black, and the amino acid in the box is a conservative sequence for formation of dimer"

Fig. 5

Phylogenetic analysis of NSTs from different plant species The number 0.05 represents the evolution distance,the number above branches represent the bootstrap value. The Numbers in parentheses are gene locus names"

Fig. 6

Effects of various treatments on MsNST expression in alfalfa stems using qRT-PCR MsNST under GA3 (gibberellin3), SA (salicylic acid), PCB (paclobutrazol) treatment. Error bars represent standard error of the mean"

Fig. 7

Identification and phenotypic analysis of transgenic Arabidopsis thaliana A: Phenotype of five-week old transgenic Arabidopsis in green house,bars=2cm; B: Ampification of MsNST gene from transgenic Arabidopsis; C: The expression level of MsNST in transgenic Arabidopsis; D: Over expression MsNST result in growth inhibition of Arabidopsis hypocotyls. n=30,error bars replace ± standard deviation. “*” or “**” on behalf of P value < 0.05 or <0.01 by stutent t test analysis; E: Statistics analysis of plant height and fresh weight of transgenic Arabidopsis thaliana and WT"

Fig. 8

Effects of overexpression MsNST on cell wall thickness, lignin, cellulose and total sugar content in transgenic Arabidopsis thaliana A: Stem cell walls of Arabidopsis stain by phloroglucinol-hydrochloric acid; B: Statistical analysis of interfascicular fiber cell wall thickness; C: Determination of lignin content of transgenic Arabidopsis stem; D: Determination of cellulose content in transgenic Arabidopsis stem; E: Determination of total sugar content in transgenic Arabidopsis stem"

Fig. 9

Expression analysis of genes related to cellulose and lignin synthesis in overexpression MsNST transgenic Arabidopsis thaliana"

Fig. 10

Identification of transgenic alfalfa and analysis of genes related to cellulose and lignin synthesis of transgenic lines A: The phenotype of overexpression alfalfa; B: Analysis of MsNST expression level of transgenic alfalfa and control; C: The plant height of overexpressing alfalfa compare with control; D: The length of six stem internodes from top; E: Relative expression levels of genes related to cellulose and lignin synthesis in transgenic alfalfa (OE1) "

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