Crops ›› 2020, Vol. 36 ›› Issue (4): 114-120.doi: 10.16035/j.issn.1001-7283.2020.04.016

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Screening of Strongly Expressed Promoters in Immature Maize Kernels

Wang Li1,2(), Wang Zuoping2(), Zhang Zhongbao2, Bai Ling1(), Wu Zhongyi2()   

  1. 1State Key Laboratory of Crop Stress Adaption and Improvement, Henan University, Kaifeng 475004, Henan, 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:2020-01-02 Revised:2020-03-05 Online:2020-08-15 Published:2020-08-11
  • Contact: Bai Ling,Wu Zhongyi E-mail:15537870386@163.com;wangzuoping@baafs.net.cn;bailing@henu.edu.cn;zwu22@126.com

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Abstract:

Promoters play an important role in regulating gene expression. To screen strongly expressed promoters in immature maize kernels, six promoters, pCaMV35SD, pUbiquitin, pZmActin1, pZmSTK2, pZm66589, and pZmbHLH148, were for EGFP expression in this study. Maize protoplasts were transfected to quickly verify the vector function. At the same time, six expression vectors were introduced into Zheng58 by pollen magnetic transfection. Later, the expression of EGFP driven by different promoters in immature maize kernels were observed 48h after pollination. The results showed that the expression of EGFP was detected in case of all six promoters but the fluorescence of EGFP driven by pCaMV35SD promoter was the strongest, and the order of EGFP fluorescence strength driven by six promoters was pCaMV35SD > pZmSTK2 >pZm66589 > pZmbHLH148 > pUbiquitin > pZmActin1, with the fluorescence detection rate of 27.17%, 27.17%, 29.83%, 23.84%, 13.40% and 30.57%, respectively.

Key words: Promoter, Pollen magnetofection, Maize kernel, Green fluorescent protein, Maize protoplast

Table 1

Basic information of promoters in this study"

启动子
Promoter		 长度
Length (bp)		 表达类型
Expression type		 参考文献
Reference
pCaMV35SD 770 组成型表达 [5]
pZmActin1 1 210 组成型表达 [6]
pUbiquitin 2 014 组成型表达 [7]
pZmSTK2 2 067 花粉特异性表达 [14]
pZm66589 2 000 胚特异性表达 [20]
pZmbHLH148 1 512 胚特异性表达 [21]

Table 2

Primers used in this study"

引物Primer 引物序列（5′-3′）Primer sequence (5′-3′)
Bar-F CTCTAGAAATGAGCCCAGAACGACGC (XbaⅠ)
Bar-R CGGATCCAGATCTTGGCAGGATATA (BamHⅠ)
pUbiquitin-EGFP-F ATCCTGCCAAGATCTGGATCCCGGTCGTGCCCCTCTCTAG
pUbiquitin-EGFP-R GCCCTTGCTCACCATGGTACCCTGCAGAAGTAACACCAAACAACAG
EGFP-F CCGGCATGCAAGCTGATAACG
EGFP-R GACACGCTGAACTTGTGGCC

Fig.1

Construction flow chart of six promoter-EGFP expression vectors a. T-DNA region of pYBA1132; b. T-DNA region of pYBA1132-Bar-EGFP; c. T-DNA region of pYBA1132-Bar-EGFP(-NptII); d. T-DNA region of pCaMV35SD driving EGFP; e. pZmActin1, pZmSTK2, pZm66589, pZmbHLH148 and pUbiquitin represent the sequences of the other five promoters, respectively, which can drive the expression of EGFP, and their T-DNA region are similar to pCaMV35SD in the Fig. 1d"

Fig.2

Enzyme digestion of six promoter-EGFP expression vectors Marker: Trans 15 000bp DNA ladder"

Fig.3

Subcellular localization of different promoters driving EGFP in protoplast EGFP: green fluorescence field; Bright: light field; Merged: superposition of light field and green fluorescence field; CK: protoplast of Zheng 58 without plasmid transfection; pCaMV35S: protoplast transferred with pYBA1132; pCaMV35SD, pZmActin1, pZmSTK2, pZm66589, pZmbHLH148 and pUbiquitin: maize protoplast transfected with EGFP recombinant vectors driven by six promoters; Bar=5μm, the same below"

Fig.4

Transient expression of EGFP driven by different promoters in Zheng 58 immature kernels CK: the immature kernels with out plasmid transfection after 48 hours of pollination; MNP: immature kernels transfected with only MNP; pCaMV35SD, pZmActin1, pUbiquitin, pZmSTK2, pZmbHLH148 and pZm66589: immature kernels transfected with corresponding promoter plasmids; Bar=200μm"

Table 3

Fluorescence detection rate of Zheng 58 immature kernels transfected with six EGFP vectors at 48h after pollination respectively"

启动子Promoter 荧光检出率Fluorescence detection rate (%)
pZmActin1 30.57±12.55a
pZm66589 29.83±9.16a
pCaMV35SD 27.17±7.96ab
pZmSTK2 27.17±7.96ab
pZmbHLH148 23.84±8.27ab
pUbiquitin 13.40±5.80b
[1] 戴景瑞, 鄂立柱 . 我国玉米育种科技创新问题的几点思考. 玉米科学, 2010,18(1):1-5.
[2] Nuccio M L. Maize. New York: Humana Press, 2018.
[3] 夏江东, 夏平 . 高等植物启动子功能和结构研究进展. 楚雄师范学院学报, 2005,20(3):41-48.
[4] 朱丽萍, 于壮, 邹翠霞 , 等. 植物逆境相关启动子及功能. 遗传, 2010,32(3):229-234.
[5] Odell J T, Nagy F, Chua N H . Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature, 1985,313(28):810-812.
[6] Mc Elroy D, Zhang W G, Cao J , et al. Isolation of an efficient actin promoter for use in rice transformation. The Plant Cell, 1990,2(2):163-171.
[7] Toki S, Takamatsu S, Nojiri C , et al. Expression of a maize ubiquitin gene promoter-bar chimeric gene in transgenic rice plants. Plant Physiology, 1992,100(3):1503-1507.
[8] Bhullar S, Chakravarthy S, Advani S , et al. Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping. Plant Physiology, 2003,132(2):988-998.
[9] Bénédicte C, Scollan C, Ross S , et al. Co-silencing of homologous transgenes in tobacco. Molecular Breeding, 2000,6(4):407-419.
[10] Xu L, Ye R J, Zheng Y S , et al. Isolation of the endosperm-specific LPAAT gene promoter from coconut (Cocos nucifera L.) and its functional analysis in transgenic rice plants. Plant Cell Reports, 2010,29(9):1061-1068.
[11] Ye R J, Zhou F, Lin Y J . Two novel positive cis-regulatory elements involved in green tissue-specific promoter activity in rice (Oryza sativa L. ssp.). Plant Cell Reports, 2012,31(7):1159-1172.
[12] Chen L, Jiang B J, Wu C X , et al. GmPRP2 promoter drives root-preferential expression in transgenic Arabidopsis and soybean hairy roots. BMC Plant Biology, 2014,14(1):245-257.
[13] Mande X, Yan L, Zhao Z Q , et al. Isolation and characterization of a green-tissue promoter from common wild rice (Oryza rufipogon Griff.). International Journal of Molecular Sciences, 2018,19(7):2009-2021.
[14] Wang H, Fan M X, Wang G H , et al. Isolation and characterization of a novel pollen-specific promoter in maize (Zea mays L.). Genome, 2017,60(6):485-495.
[15] Xu W Z, Liu W S, Ye R J , et al. A profilin gene promoter from switchgrass (Panicum virgatum L.) directs strong and specific transgene expression to vascular bundles in rice. Plant Cell Reports, 2018,37(4):1-11.
[16] Komarnytsky S, Borisjuk N . Functional analysis of promoter elements in plants. Genetic Engineering, 2003,25:113-141.
[17] Roy S, Choudhury S R, Singh S K , et al. Functional analysis of light-regulated promoter region of AtPolλ gene. Planta, 2012,235(2):411-432.
[18] Lee S C, Kim S H, Kim S R . Drought inducible OsDhn1 promoter is activated by OsDREB1A and OsDREB1D. Journal of Plant Biology, 2013,56(2):115-121.
[19] Tao Y, Wang F T, Jia D M , et al. Cloning and functional analysis of the promoter of a stress-inducible gene (ZmRXO1) in Maize. Plant Molecular Biology Reporter, 2015,33(2):200-208.
[20] Liu X Q, Tian J, Zhou X J , et al. Identification and characterization of promoters specifically and strongly expressed in maize embryos. Plant Biotechnology Journal, 2014,12(9):1286-1296.
[21] Lu X D, Chen D J, Shu D F , et al. The differential transcription network between embryo and endosperm in the early developing maize seed. Plant Physiology, 2013,162(1):440-455.
[22] Yoo S D, Cho Y H, Sheen J . Arabidopsis mesophyll protoplasts:a versatile cell system for transient gene expression analysis. Nature Protocols, 2007,2(7):1565-1572.
[23] Zhao X, Meng Z G, Wang Y , et al. Pollen magnetofection for genetic modification with magnetic nanoparticles as gene carriers. Nature Plants, 2017,3(12):956-964.
[24] Kausch A P, Owen T P, Zachwieja S J , et al. Mesophyll-specific,light and metabolic regulation of the C4 PPCZm1 promoter in transgenic maize. Plant Molecular Biology, 2001,45(1):1-15.
[25] José-Estanyol M, Pérez P, Puigdomènech P . Expression of the promoter of HyPRP,an embryo-specific gene from Zea mays in maize and tobacco transgenic plants. Gene, 2005,356(1):146-152.
[26] Chen X P, Wang Z Y, Gu R L , et al. Isolation of the maize Zpu1 gene promoter and its functional analysis in transgenic tobacco plants. Plant Cell Reports, 2007,26(9):1555-1565.
[27] Srilunchang K O, Krohn N G, Dresselhaus T . DiSUMO-like DSUL is required for nuclei positioning,cell specification and viability during female gametophyte maturation in maize. Development, 2010,137(2):333-345.
[28] 王昌涛, 梁粤, 王欢 , 等. 玉米Ubiquitin启动子的克隆及功能鉴定. 沈阳农业大学学报, 2006,37(1):9-12.
[29] 焦勇, 柳小庆, 江海洋 , 等. 植物组织特异性启动子研究进展. 中国农业科技导报, 2019,21(1):24-34.
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