Gene Cloning and Expression Analysis of Transcription Factor NAC78 in Maize

doi:10.13560/j.cnki.biotech.bull.1985.2020-1155

Abstract

Abstract:

NAC（NAM,ATAF1/2,CUC2）family transcription factors play an important role in regulating plant growth and development,responding to stress and hormone signal transduction. Exploring their functions will provide bases for analyzing the stress response mechanism in maize. The CDS sequence of the NAC78 was cloned by RT-PCR,the characteristics of the NAC78 were analyzed by bioinformatics method,and the expression patterns of NAC78 in different tissues and its responses to stresses were detected by real-time quantitative PCR. The length of NAC78’s open reading frame was 1 830 bp,encoding 609 amino acids. Its molecular weight was 67.45 kD,and isoelectric point was 5.19. The N-terminal of the NAC78 protein contained a conserved NAM domain,consisting of 8 α-helices and 7 β-sheets. The results from phylogenetic tree analysis showed that NAC78 was most closely related to the NAC86 in Setaria italica. The results of real-time quantitative PCR showed that NAC78 expressed in all tissues of maize,and the expression in the endosperm was significantly higher than that in other tissues;the expression of NAC78 increased first and then decreased,and the relative expression increased by 52.52 times compared with that on the day 5. The expression of NAC78 was induced by ABA,NaCl and PEG,which increased first and then decreased under three stress treatments,and the relative expression in the seeds increased by 11.4 times after 6 h compared with that of 0 h under ABA treatment,and the those in the leaves treated with NaCl and PEG increased by 1.87 and 2.69 times after 6 h,respectively. In sum,the expression of NAC78 in the endosperm of maize is significantly higher than that in other tissues,and presents varied responses to ABA,NaCl and PEG treatment.

Key words: NAC, transcription factor, abiotic stress, ABA

HE Shan, WANG Zhi-kang, YANG Yang, MU Yue-wei, HUANG Yu-bi, YU Guo-wu. Gene Cloning and Expression Analysis of Transcription Factor NAC78 in Maize[J]. Biotechnology Bulletin, 2021, 37(5): 1-10.

Figures/Tables 8

Table 1 Primers used in the experiment

引物名称Primer name	引物序列Primer sequence（5'-3'）
NAC78-F	ATGGCGCCGGTCAGTTTGCCTC
NAC78-R	TCACTCACTGCCGCTATTTC
NAC78-RT-F	GGCCGTTAGGTTGGTTGAGA
NAC78-RT-R	TGAAGCCGCACACCTCTAAG
Actin-F	TCACTACGACTGCCGAGCGAG
Actin-R	GAGCCACCACTGAGGACAACATTAC

Fig.1 Bioinformatics analysis of NAC78 protein A: Prediction of hydrophilicity and hydrophobicity of NAC78 protein. B: NAC78 protein domain. C: Three dimensional structure prediction of NAC78 protein

Fig.2 Multiple sequence alignment of NAC78 homologous genes in different species Sorghun bicolor (SORBI_3001G529200), Setaria italica (SETIT_034541mg), Zea mays (Zm00001d027395), Oryza sativa japonica group (Os03t0119966), Triticum aestivum (Traes_CS5A02G519300), Musa acuminata (GSMUA_Achr1T02820), Populus trichocarpa (POPTR_017G139500v3), Manihot esculenta (MANES_02G081200), Gossypium raimondii (B456_007G188800), Solanum tuberosum (XP_015165583.1), Solanum lycopersicum (Solyc02g036430), Nicotiana attenuata (NW_017670850.1), Actinidia chinensis (CEY00_Acc24239), Helianthus annuus (NC_035448.1), Cucumis sativus (Csa_5G324000), Brassica napus (BnaA05g22540D), Arabidopsis thaliana (AT5G17260), Physcomitrella patens (Pp3c8_6080V3), Selaginella moellendorffii (SELMODRAFT_100973), Marchantia polymorpha (OAE31278.1). A-E: Five subdomains of NAC domain; squiggles represents α-helices, arrows represents β-sheet and TT letters represents turns

Fig.3 Evolutionary analysis of NAC78 homologous genes

Fig.4 Relative expression of NAC78 in different tissues (A) and endosperm at different stages after pollination (B) The error line represents the standard deviation, n=3

Table 2 Cis-elements in the promoter of NAC78

元件名称Element name	序列Sequence	功能Function	数量Number	分类Classification
ABRE	ACGTG;TACGTG;CACGTA	响应脱落酸	4	激素响应元件
ABRE3a	TACGTG	响应脱落酸	1
ABRE4	CACGTA	响应脱落酸	1
CGTCA-motif	CGTCA	响应茉莉酸	3
GARE-motif	TCTGTTG	响应赤霉素	1
TGA-box	TGACGTAA	响应生长素	1
TGACG-motif	TGACG	响应茉莉酸	3
G-Box	TACGTG	响应光照	3	响应光照
GT1-motif	TACGTG	响应光照	1
Sp1	GGGCGG	响应光照	1
MBS	CAACTG	MYB转录因子结合位点	2
MYB	CAACCA;TAACCA;CCGTTG;TAACCA	MYB转录因子结合位点	7	转录因子结合位点
O2-site	GATGACATGG	O2转录因子结合位点	1
CCAAT-box	CAACGG	MYBHv1转录因子结合位点	2
RY-element	CATGCATG	调控种子发育	3	调控植物发育
ARE	TGCTATCCG	响应缺氧条件	1	响应非生物胁迫
GC-motif	CCCCCG	响应缺氧条件	2	响应非生物胁迫
TATA-box	TATATA	转录起始必须	4	转录必须元件
CAAT-box	CAAT;CAAAT	转录增强元件	4	转录必须元件

Fig.5 Effect of ABA treatment on NAC78 expression in maize kernel The error line refers to the standard deviation. sterisk refers to significant difference (*P<0.05, **P<0.01)

Fig.6 Effects of NaCl and PEG treatment on NAC78 expression in leaves The error line represents the standard deviation; sterisk represents significant difference between 0 h. (*P<0.05, **P<0.01）

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