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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (6): 1197-1202.doi: 10.3724/SP.J.1006.2021.03037

• RESEARCH NOTES • Previous Articles    

Transformation and molecular identification of maize phytochrome A1 gene (ZmPHYA1) in cotton

MA Yan-Bin1(), WANG Xia2, LI Huan-Li1, WANG Pin3, ZHANG Jian-Cheng1, WEN Jin1, WANG Xin-Sheng1, SONG Mei-Fang3, WU Xia1,*(), YANG Jian-Ping4,*()   

  1. 1Institute of Cotton Research, Shanxi Agricultural University (Shanxi Academy of Agricultural Sciences), Yuncheng 044000, Shanxi, China
    2Department of Life Science, Yuncheng University, Yuncheng 044000, Shanxi, China
    3Beijing Radiation Center, Beijing 100875, China
    4College of Agronomy, Henan Agriculture University, Zhengzhou 450002, Henan, China
  • Received:2020-06-15 Accepted:2020-11-13 Online:2021-06-12 Published:2020-12-28
  • Contact: WU Xia,YANG Jian-Ping E-mail:myb0517@163.com;wuxiab8270@126.com;jpyang@henau.edu.cn
  • Supported by:
    The Shanxi Province Key Research and Development Program(201703D211007-3);The National Natural Science Foundation of China(31201253);The National Natural Science Foundation of China(31871709);The National Major Project for Developing New GM Crops(2016ZX08010-003);The Basic Research Program of Agricultural Sciences of Shanxi Academy(YGJPY2007);The Doctoral Research Project of Yuncheng College(YQ-2017 008)

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Abstract:

In order to evaluate the potential value of maize phytochrome A1 gene (ZmPHYA1) in the improvement of cotton germplasm resources, we transferred it into upland cotton (Gossypium hirsutum L.) R15 via Agrobacterium tumefaciens-mediated transformation with glufosinate-resistance gene as selection marker. The regenerated cotton plants were obtained through callus induction, antibiotic resistance screening and differentiation induction. After screening the regenerated plants by the herbicide glufosinate ammonium in the field, PCR detection confirmed that both the target bands, including 256 bp of the glufosinate gene and 217 bp band of ZmPHYA1 gene, were detected in the homozygous transgenic plants. In addition, the exogenous ZmPHYA1 protein of about 170 kD was also checked by immuno-blot in three transgenic cotton lines. The results showed that the specific proteins could be detected in different tissues, including leaves, flowers and stems in the transgenic Line 9. The plant height of transgenic Line 9, Line 14, Line 41 were significantly shorter than that of the wild type, while the differences of other yield-related agronomic traits were not observed between the transgenic lines and the wild type. In this study, new cotton germplasms with glufosinate resistance and ZmPHYA1 gene were successfully obtained, which provided a material source for further utilization of phytochrome gene to innovate germplasm resources.

Key words: Gossypium hirsutum, maize phytochrome A1, protein expression, glufosinate resistance

Table 1

PCR primers of objective genes in transgenic cotton"

引物
Primers		 序列
Primer sequences (5′-3′)		 片段长度
Expected size (bp)
Basta-F ACCATCGTCAACCACTACATC 256
Basta-R GCTGCCAGAAA CCCACGTCAT
ZmPHYA1-1641F CAGCAGAAGGATGCACCCTAGGCTG 217
ZmPHYA1-1857R CGCTTGCAGTTCGGCGAGCCCATCA

Fig. 1

Transformation of exogenous ZmPHYA1 gene and the PCR detection of glufosinate-resistance genes in cotton plants A: callus screening and induction of cotton hypocotyl after Agrobacterium infection; B: callus induction and proliferation; C: somatic embyos formed from embryonic callus; D: regeneration plants; E: survived regenerated graft plants; F: PCR detection of glufosinate-resistance gene in some regenerated plants. Among them, Lane 1, 2 and 4 denote Line 9, Line 14, and Line 41, respectively, 3 denotes the non-transgenic regenerated plant, “-” denotes WT control, and “+” denotes plasmid positive amplification. M: DNA marker."

Fig. 2

Glufosinate resistant identification and PCR detection of exogenous ZmPHYA1 gene in cotton A, B: different positive plant offspring detected for herbicide resistance; C: isolated offspring of non-resistant plants; D: PCR detection of ZmPHYA1 in some resistant plants. Among them, “+” denotes plasmid positive amplification, 1 and 2 denote single progeny plant of Line 9, 3 and 4 denote single progeny plant of Line 14, 5 and 6 denote single progeny plant of Line 41. “-” denotes WT; M: DNA marker."

Fig. 3

Immuno-blot detection of exogenous ZmPHYA1 protein expression in transgenic cotton lines M: protein markers; WT: transgenic cotton receptor R15; non-transgenic line: non-resistant plants isolated from transgenic lines. Lane #9, #14, #41 represent Line 9, Line 14 and Line 41 of transformed exogenous ZmPHYA1 gene plants. “+” is positive cotton plant using the homologous gene ZmPHYA2 (3393 bp) in the transformed maize."

Fig. 4

Comparison of different agronomic traits in transgenic plants A: plant height phenotype of transgenic lines, Bar = 10 cm; B is the fiber length diagram of Line 9, Line 14 and Line 41, respectively, Bar = 1 cm. Histogram of C, D, E, and F represent the plant height, average weight of single boll seed cotton, lint percentage and fiber length of control (WT), Line 9, Line 14, and Line 41 lines, respectively. * indicates significant differences at P < 0.05."

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