Welcome to Acta Agronomica Sinica，

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

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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)

RichHTML380

15

PDF

497

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."

[1] Lu Z, Liu D, Liu S. Two rice cytosolic ascorbate peroxidase differentially improve salt tolerance in transgenic Arabidopsis. Plant Cell Rep, 2008,26:1909-1917.
doi: 10.1007/s00299-007-0395-7 pmid: 17571267
[2] Wang C, Yang C P, Wang Y C. Cloning and expression analysis of an APX gene from Betula platyphylla. J Northeast Agric Univ, 2009,37:79-88.
[3] 张保青, 邵敏, 黄玉新, 黄杏, 宋修鹏, 陈虎, 王盛, 谭秦亮, 杨丽涛, 李杨瑞. 甘蔗抗坏血酸过氧化物酶基因ScAPX1的克隆和表达分析. 生物技术通报, 2019,35:31-37.
Zhang B Q, Shao M, Huang Y X, Huang X, Song X P, Chen H, Wang S, Tan Q L, Yang L T, Li Y R. Cloning and expression analysis of sugarcane ascorbate peroxidase gene ScAPX1. Biotechnol Bull, 2019,35:31-37 (in Chinese with English abstract).
[4] Shia W M, Muramotoa Y, Uedaa A, Takabea T. Cloning of peroxisomal ascorbate peroxidase gene from barley and enhanced thermotolerance by overexpressing in Arabidopsis thaliana. Gene, 2001,273:23-27.
[5] Shafi A, Gill T, Zahoor I, Ahuja P S, Sreenivasulu Y, Kumar S, Singh K. Ectopic expression of SOD andAPX genes in Arabidopsis alters metabolic pools and genes related to secondary cell wall cellulose biosynthesis and improve salt tolerance. Mol Biol Rep, 2019,46:1985-2002.
doi: 10.1007/s11033-019-04648-3 pmid: 30706357
[6] 王晗, 刘涛, 何红红, 梁国平, 马宗桓, 毛娟, 陈佰鸿. 葡萄APX基因家族的鉴定与表达分析. 果树学报, 2020,37:472-484.
Wang H, Liu T, He H H, Liang G P, Ma Z H, Mao J, Chen B H. Identification and expression analysis of APX gene family in grape. J Fruit Sci, 2020,37:472-484 (in Chinese with English abstract).
[7] Cunha J R, Carvalho Fabrício E L, Lima-Neto M C, Jardim- Messeder D, Cerqueira João V A, Martins M O, Fontenele A V, Márgis-Pinheiro M, Komatsu S, Silveira Joaquim A G. Proteomic and physiological approaches reveal new insights for uncover the role of rice thylakoidalAPX in response to drought stress. J Proteomics, 2019,192:125-136.
pmid: 30170113
[8] 舒英杰, 陶源, 王爽. 高等植物种子活力的生物学研究进展. 西北植物学报, 2013,33:1709-1716.
Shu Y J, Tao Y, Wang S. Advances in biological research on seed vigor of higher plants. Acta Bot Boreali-Occident Sin, 2013,33:1709-1716 (in Chinese with English abstract).
[9] 赵昌平. 中国杂交小麦研究现状与趋势. 中国农业科技导报, 2010,12:5-8.
Zhao C P. Status and trends of hybrid wheat research in China. J Agric Sci Technol, 2010,12:5-8 (in Chinese with English abstract).
[10] 张自阳, 姜小苓, 茹振钢, 李淦, 刘明久. 人工老化对杂交小麦种子生理特性和种子活力变化的影响. 江苏农业科学, 2013,41(2):81-83.
Zhang Z Y, Jiang X L, Ru Z G, Li G, Liu M J. Effects of artificial aging on physiological characteristics and seed vigor of hybrid wheat. Jiangsu Agric Sci, 2013,41(2):81-83 (in Chinese with English abstract).
[11] 宋松泉. 种子生物学研究指南. 北京: 科学出版社, 2005. pp 92-93(in Chinese).
Song S Q. Guide to Seed Biology Research, Beijing: Science Press, 2005. pp 92-93(in Chinese).
[12] 耿晓丽, 藏新山, 王飞, 张丽媛, 田雪军, 倪中福, 姚颖垠, 胡兆荣, 辛明明, 彭惠茹, 孙其信. 小麦耐热相关转录因子基因TabZIP28的分离及功能分析. 农业生物技术学报, 2016,24:157.
Geng X L, Zang X S, Wang F, Zhang L Y, Tian X J, Ni Z F, Yao Y G, Hu Z R, Xin M M, Peng H R, Sun Q X. Isolation and functional analysis of wheat heat resistant transcription factor gene TabZIP28. J Agric Biotechnol, 2016,21:157 (in Chinese with English abstract).
[13] 刘伟灿. 干旱胁迫下大豆miR396基因家族表达分析及功能鉴定. 吉林农业大学博士学位论文, 吉林长春, 2016.
Liu W C. Expression Analysis and Functional Identification of Soybean miR396 Gene Family under Drought Stress. PhD Dissertation of Jilin Agricultural University, Changchun, Jilin, China, 2016 (in Chinese with English abstract).
[14] Yu Y H, Ni Z Y, Wang Y, Wan H N, Hu Z, Jiang Q Y, Sun X J, Zhang H. Overexpression of soybean miR169c confers increased drought stress sensitivity in transgenic Arabidopsis thaliana. Plant Sci, 2019,285:68-78.
pmid: 31203895
[15] 何小梦. miR166和miR319在黄瓜响应盐胁迫中的功能分析. 华中农业大学硕士学位论文, 湖北武汉, 2019.
He X M. Functional Analysis of miR166 and miR319 in Cucumber Response to Salt Stress. MS Thesis of Huazhong Agricultural University, Wuhan, Hubei, China, 2019 (in Chinese with English abstract).
[16] 宋超. 水稻种子人工老化过程中抗氧化系统的变化. 中国农业科学院研究生院硕士学位论文, 北京, 2012.
Song C. Changes of Antioxidant System during Artificial Aging of Rice Seeds. MS Thesis of Graduate School of Chinese Academy of Agricultural Sciences, Beijing, China, 2012 (in Chinese with English abstract).
[17] 刘建军, 马俊华, 孟俊文, 王自霞, 周小梅. 玉米种子老化过程中抗氧化酶活性的变化. 山西农业科学, 2013,41:907-910.
Liu J J, Ma J H, Meng J W, Wang Z X, Zhou X M. Changes of antioxidant enzyme activity in maize seed aging. J Shanxi Agric Sci, 2013,41:907-910 (in Chinese with English abstract).
[18] 周洁. microRNA 167基因在母系控制胚胎和种子发育中的重要作用. 上海师范大学硕士学位论文, 上海, 2019.
Zhou J. microRNA 167 Plays an Important Role in Maternal Control of Embryo and Seed Development. MS Thesis of Shanghai Normal University, Shanghai, China, 2019 (in Chinese with English abstract).
[19] Jasdeep C P, Harinder V, Koushik B, Rahul S J, Gyanendra P S. Molecular cloning and in-silico characterization of high temperature stress responsivepAPX gene isolated from heat tolerant Indian wheat cv. Raj 3765. BMC Res Notes, 2014,7:7137.
[20] 王爱君. 芥菜种子超干处理及老化的生理生化特性研究. 西南大学硕士学位论文, 重庆, 2011.
Wang A J. Study on Physiological and Biochemical Characteristics of Mustard Seeds Treated with Super-Drying and Aging. MS Thesis of Southwest University, Chongqing, China, 2011 (in Chinese with English abstract).
[21] 王玉红. 高羊茅、老芒麦、燕麦种子劣变与膜结构和透性关系的研究. 中国农业大学硕士学位论文, 北京, 2008.
Wang Y H. Study on the Relationship between Seed Deterioration and Membrane Structure and Permeability of Tall Fesco, Old Mann-Wheat and Oat. MS Thesis of China Agricultural University, Beijing, China, 2008 (in Chinese with English abstract).
[22] 杨江伟. 马铃薯干旱相关microRNA的鉴定及功能研究. 甘肃农业大学博士学位论文, 甘肃兰州, 2015.
Yang J W. Identification and Functional Study of Drought- Related microRNA in Potato. PhD Dissertation of Gansu Agricultural University, Lanzhou, Gansu, China, 2015 (in Chinese with English abstract).
[23] 龚淑敏. 甜玉米种子活力相关microRNA的鉴定与分析. 中国计量学院硕士学位论文, 浙江杭州, 2015.
Gong S M. Identification and Analysis of microRNA Related to Seed Vigor of Sweet Corn. MS Thesis of China Jiliang University, Hangzhou, Zhejiang, China, 2015 (in Chinese with English abstract with English abstract).
[1] CUI Lian-Hua, ZHAN Wei-Min, YANG Lu-Hao, WANG Shao-Ci, MA Wen-Qi, JIANG Liang-Liang, ZHANG Yan-Pei, YANG Jian-Ping, YANG Qing-Hua. Molecular cloning of two maize (Zea mays) ZmCOP1 genes and their transcription abundances in response to different light treatments [J]. Acta Agronomica Sinica, 2022, 48(6): 1312-1324.
[2] CHEN Song-Yu, DING Yi-Juan, SUN Jun-Ming, HUANG Deng-Wen, YANG Nan, DAI Yu-Han, WAN Hua-Fang, QIAN Wei. Genome-wide identification of BnCNGC and the gene expression analysis in Brassica napus challenged with Sclerotinia sclerotiorum and PEG-simulated drought [J]. Acta Agronomica Sinica, 2022, 48(6): 1357-1371.
[3] ZHOU Hui-Wen, QIU Li-Hang, HUANG Xing, LI Qiang, CHEN Rong-Fa, FAN Ye-Geng, LUO Han-Min, YAN Hai-Feng, WENG Meng-Ling, ZHOU Zhong-Feng, WU Jian-Ming. Cloning and functional analysis of ScGA20ox1 gibberellin oxidase gene in sugarcane [J]. Acta Agronomica Sinica, 2022, 48(4): 1017-1026.
[4] JIN Min-Shan, QU Rui-Fang, LI Hong-Ying, HAN Yan-Qing, MA Fang-Fang, HAN Yuan-Huai, XING Guo-Fang. Identification of sugar transporter gene family SiSTPs in foxtail millet and its participation in stress response [J]. Acta Agronomica Sinica, 2022, 48(4): 825-839.
[5] XIE Qin-Qin, ZUO Tong-Hong, HU Deng-Ke, LIU Qian-Ying, ZHANG Yi-Zhong, ZHANG He-Cui, ZENG Wen-Yi, YUAN Chong-Mo, ZHU Li-Quan. Molecular cloning and expression analysis of BoPUB9 in self-incompatibility Brassica oleracea [J]. Acta Agronomica Sinica, 2022, 48(1): 108-120.
[6] YIN Ming, YANG Da-Wei, TANG Hui-Juan, PAN Gen, LI De-Fang, ZHAO Li-Ning, HUANG Si-Qi. Genome-wide identification of GRAS transcription factor and expression analysis in response to cadmium stresses in hemp (Cannabis sativa L.) [J]. Acta Agronomica Sinica, 2021, 47(6): 1054-1069.
[7] TANG Rui-Min, JIA Xiao-Yun, ZHU Wen-Jiao, YIN Jing-Ming, YANG Qing. Cloning of potato heat shock transcription factor StHsfA3 gene and its functional analysis in heat tolerance [J]. Acta Agronomica Sinica, 2021, 47(4): 672-683.
[8] JIA Xiao-Ping, LI Jian-Feng, ZHANG Bo, QUAN Jian-Zhang, WANG Yong-Fang, ZHAO Yuan, ZHANG Xiao-Mei, WANG Zhen-Shan, SANG Lu-Man, DONG Zhi-Ping. Responsive features of SiPRR37 to photoperiod and temperature, abiotic stress and identification of its favourable allelic variations in foxtail millet (Setaria italica L.) [J]. Acta Agronomica Sinica, 2021, 47(4): 638-649.
[9] YANG Qin-Li, YANG Duo-Feng, DING Lin-Yun, ZHANG Ting, ZHANG Jun, MEI Huan, HUANG Chu-Jun, GAO Yang, YE Li, GAO Meng-Tao, YAN Sun-Yi, ZHANG Tian-Zhen, HU Yan. Identification of a cotton flower organ mutant 182-9 and cloning of candidate genes [J]. Acta Agronomica Sinica, 2021, 47(10): 1854-1862.
[10] HE Xiao, LIU Xing, XIN Zheng-Qi, XIE Hai-Yan, XIN Yu-Feng, WU Neng-Biao. Molecular cloning, expression, and enzyme kinetic analysis of a phenylalanine ammonia-lyase gene in Pinellia ternate [J]. Acta Agronomica Sinica, 2021, 47(10): 1941-1952.
[11] LI Guo-Ji, ZHU Lin, CAO Jin-Shan, WANG You-Ning. Cloning and functional analysis of GmNRT1.2a and GmNRT1.2b in soybean [J]. Acta Agronomica Sinica, 2020, 46(7): 1025-1032.
[12] Jin-Feng ZHAO,Yan-Wei DU,Gao-Hong WANG,Yan-Fang LI,Gen-You ZHAO,Zhen-Hua WANG,Yu-Wen WANG,Ai-Li YU. Identification of PEPC genes from foxtail millet and its response to abiotic stress [J]. Acta Agronomica Sinica, 2020, 46(5): 700-711.
[13] LIANG Si-Wei,JIANG Hao-Liang,ZHAI Li-Hong,WAN Xiao-Rong,LI Xiao-Qin,JIANG Feng,SUN Wei. Genome-wide identification and expression analysis of HD-ZIP I subfamily genes in maize [J]. Acta Agronomica Sinica, 2020, 46(4): 532-543.
[14] Tong-Hong ZUO, He-Cui ZHANG, Qian-Ying LIU, Xiao-Ping LIAN, Qin-Qin XIE, Deng-Ke HU, Yi-Zhong ZHANG, Yu-Kui WANG, Xiao-Jing BAI, Li-Quan ZHU. Molecular cloning and expression analysis of BoGSTL21 in self-incompatibility Brasscia oleracea [J]. Acta Agronomica Sinica, 2020, 46(12): 1850-1861.
[15] WANG Yan-Hua,XIE Ling,YANG Bo,CAO Yan-Ru,LI Jia-Na. Flowering genes in oilseed rape: identification, characterization, evolutionary and expression analysis [J]. Acta Agronomica Sinica, 2019, 45(8): 1137-1145.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Add: No.12 Zhongguancun South Street, Beijing 100081,China Email:xbzw@chinajournal.net.cn
Supported by Beijing Magtech Co.Ltd