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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (3): 405-415.doi: 10.3724/SP.J.1006.2021.01049

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Cloning and potential function analysis of ascorbic peroxidase gene of hybrid wheat in seed aging

YUE Jie-Ru1,2,3(), BAI Jian-Fang2,3(), ZHANG Feng-Ting2,3, GUO Li-Ping2,3, YUAN Shao-Hua2,3, LI Yan-Mei2,3, ZHANG Sheng-Quan2,3, ZHAO Chang-Ping2,3,*(), ZHANG Li-Ping1,2,3,*()   

  1. 1College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206, China
    2Beijing Engineering and Technique Research Center for Hybrid Wheat, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
    3Municipal Key Laboratory of the Molecular Genetics of Hybrid Wheat, Beijing 100097, China
  • Received:2020-06-12 Accepted:2020-10-14 Online:2021-03-12 Published:2020-10-28
  • Contact: ZHAO Chang-Ping,ZHANG Li-Ping E-mail:1041705041@qq.com;baijianfang1313@yahoo.com;cp_zhao@vip.sohu.com;lpzhang8@126.com
  • Supported by:
    National Key Research and Development Program of China(2018YFD0100904);Beijing Excellent Talents Project;Outstanding Scientist Program of Beijing Academy of Agricultural and Forestry Sciences(JKZX201907)

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Abstract:

In this study, the seeds of two-line hybrid wheat of BS-type BS1453/11GF5135 and its parents BS1453 (female parent) and 11GF5135 (male parent) were used as materials, and the ascorbate peroxidase gene (TaAPX) was obtained by homologous cloning in BS1453/11GF5135. The gene contains an open reading frame (ORF) of 832 bp, which encoded a total of 277 amino acids. The interaction between miRNAs and TaAPX was predicted by bioinformatics analysis, and the results showed that TaAPX may be regulated by miR396 and other miRNAs that related to stress resistance and seed viability. In addition, it was found that APX protein mainly interacted with redox-related enzymes via protein interaction prediction analysis. qPCR and enzyme activity analysis were conducted on embryos of parents and hybrid at different aging time, it was found that the expression trend of TaAPX gene in hybrid and parents were first up-regulated and then down-regulated. However, the expression of TaAPX gene in hybrid seeds peaked on the seventh day, and it began to decline in parents seeds on the fifth day. There was an antagonistic effect between miR396 and TaAPX. With the increase of aging time, the activity of APX enzyme in parents seeds showed a downtrends, while it showed a temporary downtrends on the third day in hybrid seeds, then it increased and began to decline on the ninth day. It was suggested that the ability of APX enzyme to remove peroxides in the hybrid seeds was higher than that in the parents seeds under the aging condition, that is, the anti-aging ability of hybrid was higher than that of the parents. Moreover, the regulation of seed activity by TaAPX gene was a complex multi-factor regulation process. This study laid a foundation for the further research on the molecular regulation mechanism of the seed vigor of BS type hybrid wheat.

Key words: BS type hybrid wheat, artificial aging, ascorbate peroxidase, gene cloning, expression analysis

Table 1

Gene cloning of TaAPX gene and primers of qPCR used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 退火温度
Annealing
temperature (℃)		 引物用途
Primer purpose
APX-F TATCTCTAGAGGATCCATGGCGGCTCCGGT 71.37 扩增基因引物序列
Amplified gene primer sequences
APX-R TGCTCACCATGGATCCTTACTTGCTCCTCTTGGAAGCCTCG 72.27
APX-qF CAACCGTTGAGTTCATCCCTG 57.8 荧光定量PCR引物序列
RT-qPCR primer sequences
APX-qR TGGTGCGCCTTTCTTAGCAT 60
18S rRNA-qF CGCGCTACGGCTTTGACCTA 56 内参引物序列
Internal reference primer sequences
18S rRNA-qR CGGCAGATTCCCACGCGTTACG 58
miR396-qF CGGTCTTCCACAGCTTTCTT 75 荧光定量PCR引物序列
RT-qPCR primer sequences
miR396-qR GTGCAGGGTCCGAGGT 76
U6snRNA-qF CAAGGATGACACGCAAATTCG 80 内参引物序列
Internal reference primer sequences
U6snRNA-qR GTGCAGGGTCCGAGGT 82.5

Fig. 1

Grain morphology of parents and hybrids"

Fig. 2

Seed germination phenotype and germination statistics before and after artificial aging A: germination phenotypes of artificially aged and untreated seeds of hybrid and parents. B: effects of artificial aging on germination rate, root length and shoot length of hybrids and parents. ** and * indicate significant differences at the 0.01 and 0.05 probability levels, respectively. 0-5 d: Day(s) of aging treatment."

Fig. 3

Amplification products of TaAPX gene M: DL2000 DNA marker; 1: amplified product of BS1453; 2: amplified product of 11GF5135; 3: amplified product of BS1453/ 11GF5135."

Fig. 4

Multiple alignment analysis of TaAPX amino acid sequences"

Fig. 5

CDS alignment comparative analysis of parents and hybrid"

Fig. 6

Prediction of tertiary structure and phosphorylation site of TaAPX protein A: tertiary structure of TaAPX protein; B: phosphorylation site prediction of TaAPX protein."

Fig. 7

Phylogenetic analysis of TaAPX"

Fig. 8

Interaction model and site of TaAPX gene with miRNA and protein A: interaction mapping between TaAPX gene and miRNA; B: the hairpin structure diagram of miRNA; C: network model of proteins interacting with TaAPX proteins; D: the interaction site between TaAPX gene and miR396."

Fig. 9

Expression pattern analysis of TaAPX gene and miR396 in hybrid and parental seed embryos at different aging time A: the expression of TaAPX gene and miR396 in embryo of male seeds, under different aging conditions; B: the expression of TaAPX gene and miR396 in embryo of female seeds, under different aging conditions; C: the expression of TaAPX gene and miR396 in embryo of hybrid seeds, under different aging conditions. 0-9 d: day(s) of aging treatment."

Fig. 10

Analysis of APX enzyme activity in hybrids and parents at different aging times A: changes of APX enzyme activity in embryo of male seeds, under different aging conditions; B: changes of APX enzyme activity in embryo of female seeds, under different aging conditions; C: changes of APX enzyme activity in embryo of hybrid seeds, under different aging conditions. 0-9 d: day(s) of aging treatment."

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