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
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