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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (12): 2008-2016.doi: 10.3724/SP.J.1006.2020.03022

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Cloning and functional analysis of ZmbHLH161 gene in maize

Meng-Ting YANG1,2(), Chun ZHANG2(), Zuo-Ping WANG2, Hua-Wen ZOU1,*(), Zhong-Yi WU2,*()   

  1. 1College of Agriculture, Yangtze University, Jingzhou 434023, Hubei, China
    2Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences / Beijing Key Laboratory of Agricultural Gene Resources and Biotechnology, Beijing 100097, China
  • Received:2020-03-28 Accepted:2020-06-02 Online:2020-06-22 Published:2020-06-22
  • Contact: Hua-Wen ZOU,Zhong-Yi WU E-mail:yangmengting213@163.com;spring2007318@163.com;zouhuawen73@hotmail.com;zwu22@126.com
  • Supported by:
    Beijing Academy of Agricultural Youth Fund(QNJJ201724);Beijing Academy of Agricultural and Forestry Sciences(KJCX20200204);Beijing Academy of Agricultural and Forestry Sciences(KJCX20200205);Beijing Academy of Agricultural and Forestry Sciences(KJCX20200407);National Natural Science Foundation of China(31971839);National Natural Science Foundation of China(31471510)

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Abstract:

bHLH transcription factors are the second largest family of transcription factors in plants and play an important role in regulating plant growth and development, signal transduction, and stress response. In order to study the function of maize bHLH family genes in response to stress, ZmbHLH161 (AC: NC_AQK75074) gene from maize root tissue was cloned in this study. Bioinformatic analysis showed that this gene contains 3 exons, the full-length of its cDNA is 1460 bp, coding sequence is 1059 bp in length, encoding 352 amino acids. It exists as a single copy in the maize genome and its function is unknown. The molecular weight of ZmbHLH161 protein is 37.1 kD, and its theoretical isoelectric point is 6.10, with a conserved domain unique to the bHLH transcription factor family, but does not have a transmembrane structure or signal peptide. It is a hydrophilic protein, and the secondary structure of the protein has a maximum proportion of 42.05%. Transient expression experiments in maize protoplasts showed that ZmbHLH161 was localized in the nucleus. Real-time quantitative PCR (qPCR) analysis showed that under normal growth conditions, ZmbHLH161 was mainly expressed in roots and immature embryos, while under dehydration and drought treatment, ZmbHLH161 was up-regulated in maize seedling leaves. After treated with different concentrations of NaCl, the root length of ZmbHLH161 transgenic heterologous Arabidopsis strains was not significantly different from that of wild type, and their root was longer than that of wild type after being treated with different concentrations of mannitol. It is speculated that ZmbHLH161 gene may be involved in the response of maize to osmotic stress.

Key words: maize, ZmbHLH161, transcription factor, protoplast, qPCR, osmotic stress

Table 1

Primers used in this study"

引物对编号
Number of primer pairs		 引物名称
Primer name		 引物序列
Primer sequence (5′-3′)
1 pZmbHLH161-F AGCGTTAGTCACAGTTGTTGCC
pZmbHLH161-R CGTAAGCTGGTCCAATCGGTAC
2 pZmbHLH161SL-F ATGAATTCATGGCGCTCGAAG (EcoR I)
pZmbHLH161SL-R AATAAGCTTGTGGCCTGGCC (Hind III)
3 pZmbHLH161RT-F AGCATCAGCTCCTGTCCGTGTC
pZmbHLH161RT-R CTCTCCATCTCGTCCGTGTTCC
4 pZmbHLH161At-F ATGAATTCAGCGTTAGTCACAGTTGTTGCC (EcoR I)
pZmbHLH161At-R ATGGTACCTCAGTGGCCTGGCCCCCCGC (Kpn I)
5 Actin-F TACGAGATGCCTGATGGTCAGGTCA
Actin-R TGGAGTTGTACGTGGCCTCATGGAC

Fig. 1

Bioinformatics analysis of ZmbHLH161 A: amino acid sequence alignment; B: protein secondary structure prediction; C: transmembrane structure prediction."

Fig. 2

Phylogenetic tree of the deduced amino acid sequences of the bHLH transcription factors in Arabidopsis, rice, and maize"

Fig. 3

Promoter analysis of ZmbHLH161"

Fig. 4

Subcellular localization of ZmbHLH161 protein in maize leaf protoplasts GFP: GFP-labeled ZmbHLH161 subcellular localization in maize protoplasts; DAPI: DAPI-labeled protoplast nuclei; Bright: protoplasts under the same field of view light microscope; Merged: superposition of light microscope and fluorescence photos; CK: transfer empty vector protoplasts; ZmbHLH161: transfer into protoplast of target vector; Bar = 3 μm."

Fig. 5

Relative expression level of ZmbHLH161 in different maize tissues"

Fig. 6

Real-time quantitative PCR analysis for expression level of ZmbHLH161 gene under different treatments in leaf and root A: drought treatment; B: dehydration treatment; C: cold (4℃) treatment; D: high salt (200 mmol L-1 NaCl) treatment. The error bar represents ± SD of three biological replications."

Fig. 7

PCR detection of T1 generation Arabidopsis M: DL2000 marker; P: positive control; W: wild-type Arabidopsis; N: negative control; 1-3: T1 Arabidopsis generation."

Fig. 8

Root length of transgenic Arabidopsis thaliana on gradient NaCl concentrations A-D indicates the seedling growth under the NaCl concentrations of 0, 0.10, 0.15, and 0.18 mol L-1; E: the average main root length; WT: wild type; OE: ZmbHLH161 transgenic Arabidopsis thaliana. Bar = 1.5 cm"

Fig. 9

Root length of transgenic Arabidopsis thaliana on gradient mannitol concentrations A-D indicates the seedling growth under the mannitol concentrations of 0, 0.15, 0.2, and 0.3 mol L-1; E: the average main root length of Arabidopsis at different mannitol concentrations; WT: wild type; OE: ZmbHLH161 of transgenic Arabidopsis thaliana; Bar = 1.5 cm. * and ** indicate that the root length of transgenic Arabidopsis thaliana is significantly different from that of wild type (P < 0.05) and the difference is extremely significant (P<0.01)."

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