Welcome to Acta Agronomica Sinica，

Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (4): 1027-1034.doi: 10.3724/SP.J.1006.2022.14066

• RESEARCH NOTES • Previous Articles    

Creation and identification of peanut germplasm tolerant to triazolopyrimidine herbicides

LIU Jia-Xin1,2(), LAN Yu1,2, XU Qian-Yu1, LI Hong-Ye1, ZHOU Xin-Yu3, ZHAO Xuan1, GAN Yi1, LIU Hong-Bo1, ZHENG Yue-Ping1, ZHAN Yi-Hua1, ZHANG Gang3, ZHENG Zhi-Fu1,2,*()   

  1. 1College of Advanced Agricultural Sciences, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
    2College of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
    3Wendeng Jiahe Seed Corporation, Ltd., Weihai 264400, Shandong, China
  • Received:2021-04-18 Accepted:2021-07-12 Online:2022-04-12 Published:2021-08-11
  • Contact: ZHENG Zhi-Fu E-mail:252606752@qq.com;zzheng@zafu.edu.cn
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    National Natural Science Foundation of China(31871660)

RichHTML396

11

PDF

193

Abstract:

At present, the germplasm resources of herbicide-tolerant peanut (Arachis hypogaea) are scarce, which restrict the diversification of peanut-based cropping system. To create peanut germplasm with tolerance to different herbicides, a mutant population consisting of more than 55,000 peanut lines were generated by ethylmethanesulfonate mutagenesis. We screened this population with a variety of different herbicides to obtain multiple mutants with tolerance to different herbicides. One of these lines displayed strong tolerance to florasulam and flumetsulam in the experiments with foliar herbicide spraying under field conditions as well as in various laboratory evaluation for the herbicide tolerance, while the herbicide-tolerant trait did not have adverse effects on peanut yield and quality. To determine whether this trait was associated with a target-site-based resistance to the herbicides, we compared gene sequences and relative expression levels of these two acetohydroxyacid synthases (AHASs) as the herbicide target enzymes between the mutant and wild type. Molecular cloning and sequence analysis revealed that peanut chromosome A10 and B10 each contained a gene, named as AhAHAS1a and AhAHAS1b, which were highly similar to Arabidopsis AHAS. Peanut chromosome A08 and B08 also each carried an AHAS gene, named as AhAHAS2a and AhAHAS2b, respectively. However, compared with the wild type, the genes in the mutants had no nucleotide substitutions that could alter the amino acid sequences. Furthermore, it was evident that there was no significant difference in the relative expression levels of AhAHAS genes between the mutant and wild type. In summary, these results indicate that the herbicide tolerance of the mutant might be caused by non-target-site-based resistance mechanism.

Key words: Arachis hypogaea, ethylmethanesulfonate mutagenesis, triazolopyrimidines, herbicide tolerance, quality trait

Table 1

Primer sequences used in this study"

引物
Primer		 引物序列
Primer sequence (5'-3')		 目的基因
Target gene
LY1/2FP ATGGCTGCCACTGCTTCCAAAC A10或B10染色体的AHAS
AHAS on chromosome A10 or B10
LY1RP TCAATATTTTGTTCTGCCATCGCCT
LY2RP CAATATTTTGTTCTGCCATCACCT
XQY1FP CATTCTCCACCGTATCTCCATC A08或B08染色体的AHAS
AHAS on chromosome A08 and B08
XQY1RP TACATGCCACTGTCTGGTTACTACT
LY31FP ACGAAGCATGCTTACCTTGTTCTTG A10或B10染色体的AHAS
AHAS on chromosome A10 and B10
LY31RP AACATAACTGGTTGATCCCAATTGG
XQY2FP AGCTTGAGGCTTTCGCGAGT A08或B08染色体的AHAS
AHAS on chromosome A08 and B08
XQY2RP CCCTTTTCCTCCAAGATCCCA
XQY4FP CAGGATTTGCCGGTGATGATG β-肌动蛋白
β-Actin
XQY4RP TCTGTTGGCCTTCGGGTTGAG

Fig. 1

Effects of foliar spraying with herbicides on seedling growth in the wild type and mutant peanuts Peanut plants were treated with herbicides at the 3-leaf growth stage. The amount of herbicide “Mai Xi” (450 mL hm-2) used was three times that of normal field application, and photographs were taken after 4 weeks of the treatment. A: the wild type ‘Shanhua 15’; B: the herbicide tolerant mutant H2-20-1 (M7)."

Fig. 2

Effects of foliar spraying with herbicide on growth in the wild type and mutant peanut at maturation stage Peanut plants were treated with herbicides at the 3-leaf growth stage. The amount of herbicide “Mai Xi” (450 mL hm-2) used was three times that of normal field application, and photographs were taken at maturity stage. A: the wild type ‘Shanhua 15’; B: the herbicide tolerant mutant H2-20-1 (M7)."

Fig. 3

Effects of seed soaking with herbicides on root growth of mutant H2-20-1 and wild type ‘Shanhua 15’ The concentrations of flumetsulam (Flu) and florasulam (Flo) were 9.90 g hm-2 and 7.50 g hm-2, respectively. The amount of herbicides used was twice that of normal field application and photographs were taken on the 15th day after treating. WT: wild type."

Fig. 4

Effects of herbicides on root growth of mutant H2-20-1 and wild type ‘Shanhua 15’ under the condition of sand culturing The herbicides were dissolved in water and mixed into sand. The concentrations of flumetsulam (Flu) and florasulam (Flo) were 9.90 g hm-2 and 7.50 g hm-2, respectively. The amount of herbicides used was twice that of normal field application and photographs were taken on the 15th day after treating. WT: wild type."

Fig. 5

Oil content of mature seeds in wild type and peanut mutant H2-20-1 Two batches of seeds from different planting years (in 2020 and 2019) were used for analysis. WT: wild type. t-test statistical analysis revealed that there was no significant difference in seed oil content between the mutant and wild type peanut sampled in two consecutive years."

Fig. 6

Fatty acid composition of mature seeds of wild type and peanut mutant H2-20-1 Two batches of seeds from different planting years in 2020 and 2019 were used for data analysis. WT: wild type. The asterisk (*) represents significant difference at P < 0.05 compared with WT by t-test."

Fig. 7

Amino acid composition of mature seeds in wild type and peanut mutant H2-20-1 WT: wild type. The asterisk (*) represents significant statistical difference at P < 0.05 compared with WT by t-test."

Table 2

Sequence similarity of the AHAS1 between peanut mutant H2-20-1 (MT) and wild type (WT)"

AHAS1a (MT) AHAS1a (WT) AHAS1b (MT) AHAS1b (WT)
AHAS1a (MT) 100 99 99
AHAS1a (WT) 99 99
AHAS1b (MT) 100
AHAS1b (WT)

Table 3

Sequence similarity of the AHAS2 between peanut mutant H2-20-1 (MT) and wild type (WT)"

AHAS2a (MT) AHAS2a (WT) AHAS2b (MT) AHAS2b (WT)
AHAS2a (MT) 100 99 99
AHAS2a (WT) 99 99
AHAS2b (MT) 100
AHAS2b (WT)

Fig. 8

Relative expression patterns of AHAS genes in different tissues of wild-type peanut at different developmental stages Value of gene relative expression level in the stem at seeding stage was set at “1”. DAF: days after flowering."

Fig. 9

Relative expression patterns of AHAS genes between peanut mutant H2-20-1 and wild type The relative expression level of AHAS2 in wild-type leaves was set at “1”. t-test showed there was no significant difference in the relative expression level of AHAS gene between peanut mutant and wild type."

[1] 万书波. 我国花生产业面临的机遇与科技发展战略. 中国农业科技导报, 2009, 11(1):7-12.
Wan S B. Opportunities facing peanut industry in China and strategies for its science and technology development. J Agric Sci Technol, 2009, 11(1):7-12 (in Chinese with English abstract).
[2] 丁小霞, 李培武, 周海燕, 李娟, 白艺珍. 花生农药最大残留限量标准比对研究. 中国油料作物学报, 2011, 33:527-531.
Ding X X, Li P W, Zhou H Y, Li J, Bai Y Z. Comparative study on maximum residue limits standards of pesticides in peanuts. Chin J Oil Crop Sci, 2011, 33:527-531 (in Chinese with English abstract).
[3] Jabusch T W, Tjeerdema R S. Chemistry and fate of triazolopyrimidine sulfonamide herbicides. Rev Environ Contam Toxicol, 2008, 193:31-52.
doi: 10.1007/978-0-387-73163-6_2 pmid: 20614343
[4] Cui H L, Li X J, Wang G Q, Wang J P, Wei S H, Cao H Y. Acetolactate synthase proline (197) mutations confer tribenuron-methyl resistance in Capsella bursa-pastoris populations from China. Pestic Biochem Physiol, 2012, 102:229-232.
doi: 10.1016/j.pestbp.2012.01.007
[5] Lee H, Ullrich S E, Burke I C, Yenish J, Paulitz T C. Interactions between the root pathogen Rhizoctonia solani AG-8 and acetolactate-synthase-inhibiting herbicides in barley. Pest Manag Sci, 2012, 68:845-852.
doi: 10.1002/ps.v68.6
[6] Liu W, Bi Y, Li L, Yuan G, Wang J. Molecular basis of resistance to tribenuron in water starwort (Myosoton aquaticum) populations from China. Weed Sci, 2013, 61:390-395.
doi: 10.1614/WS-D-12-00200.1
[7] 徐倩玉, 兰玉, 刘嘉欣, 周新宇, 张刚, 郑志富. 乙酰羟酸合酶抑制剂类除草剂的植物抗性机制. 作物学报, 2019, 45:1295-1302.
doi: 10.3724/SP.J.1006.2019.93003
Xu Q Y, Lan Y, Liu J X, Zhou X Y, Zhang G, Zheng Z F. Mechanisms underlying plant resistance to the acetohydroxyacid synthase-inhibiting herbicides. Acta Agron Sin, 2019, 45:1295-1302 (in Chinese with English abstract).
[8] Tan S, Evans R R, Dahmer M L, Singh B K, Shaner D L. Imidazolinone-tolerant crops: history, current status and future. Pest Manag Sci, 2005, 61:246-257.
doi: 10.1002/(ISSN)1526-4998
[9] Pozniak C J, Birk I T, O’Donoughue L S, Ménard C, Hucl P J, Singh B K, Physiological and molecular characterization of mutation-derived imidazolinone resistance in spring wheat. Crop Sci, 2004, 44:1434-1443.
doi: 10.2135/cropsci2004.1434
[10] Lee H, Rustgi S, Kumar N, Burke I, Yenish J P, Gill K S, von Wettstein D, Ullrich S E. Single nucleotide mutation in the barley acetohydroxy acid synthase (AHAS) gene confers resistance to imidazolinone herbicides. Proc Natl Acad Sci USA, 2011, 108:8909-8913.
doi: 10.1073/pnas.1105612108
[11] Rajasekaran K, Grula J W, Anderson D M. Selection and charac-terization of mutant cotton (Gossypium hirsutum L.) cell lines resistant to sulfonylurea and imidazolinone herbicides. Plant Sci, 1996, 199:115-124.
[12] Ghio C, Ramos M L, Altieri E, Bulos M, Sala C A. Molecular characterization of Als1, an acetohydroxyacid synthase mutation conferring resistance to sulfonylurea herbicides in soybean. Theor Appl Genet, 2013, 126:2957-2968.
doi: 10.1007/s00122-013-2185-7
[13] Walter K L, Strachan S D, Ferry N M, Albert H H, Castle L A, Sebastian S A. Molecular and phenotypic characterization of Als1 and Als2 mutations conferring tolerance to acetolactate synthase herbicides in soybean. Pest Manag Sci, 2014, 70:1831-1839.
doi: 10.1002/ps.3725 pmid: 24425499
[14] 高建芹, 浦惠明, 戚存扣, 张洁夫, 龙卫华, 胡茂龙, 陈松, 陈新军, 陈锋, 顾慧. 抗咪唑啉酮油菜种质的发现与鉴定. 植物遗传资源学报, 2010, 11:369-373.
Gao J Q, Pu H M, Qi C K, Zhang J F, Long W H, Hu M L, Chen S, Chen X J, Chen F, Gu H. Identification of imidazoli-done- resistant oilseed rape mutant. J Plant Genet Resour, 2010, 11:369-373 (in Chinese with English abstract).
[15] Kolkman J M, Slabaugh M B, Bruniard J M, Berry S, Bushman B S, Olungu C, Maes N, Abratti G, Zambelli A, Miller J F, Leon A, Knapp S J. Acetohydroxyacid synthase mutations conferring resistance to imidazolinone or sulfonylurea herbicides in sunflower. Theor Appl Genet, 2004, 109:1147-1159.
pmid: 15309298
[16] Sala C A, Bulos M, Echarte M, Whitt S R, Ascenzi R. Molecular and biochemical characterization of an induced mutation conferring imidazolinone resistance in sunflower. Theor Appl Genet, 2008, 118:105-112.
doi: 10.1007/s00122-008-0880-6
[17] Wright T R, Penner D. Cell selection and inheritance of imidazolinone resistance in sugar beet (Beta vulgaris). Theor Appl Genet, 1998, 96:612-620.
doi: 10.1007/s001220050779
[18] 周超, 张勇, 路兴涛, 马冲, 吴翠霞, 宋敏, 张田田, 孔繁华. 8种土壤处理除草剂对花生田杂草的防除效果及安全性评价. 农药, 2019, 58(3):226-229.
Zhou C, Zhang Y, Lu X T, Ma C, Wu C X, Song M, Zhang T T, Kong F H. The control effects and safety of eight soil treatment herbicides in peanut field. Agrochem, 2019, 58(3):226-229 (in Chinese with English abstract).
[19] 唐永常, 李永超, 耿锐. 麦套花生可持续发展技术存在问题及对策. 河南农业, 2014, (19):47.
Tang Y C, Li Y C, Geng R. Problems and countermeasures of sustainable development technology of wheat-covered peanut. Henan Agric, 2014, (19):47 (in Chinese).
[20] Zheng Z, Xia Q, Dauk M, Selvaraj G, Zou J. Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3- phosphate acyltransferase gene family, is essential for tapetum differentiation and male fertility. Plant Cell, 2003, 15:1872-1887.
doi: 10.1105/tpc.012427
[21] Gan Y, Song Y, Chen Y, Liu H, Yang D, Xu Q, Zheng Z. Transcriptome analysis reveals a composite molecular map linked to unique seed oil profile of Neocinnamomum caudatum(Nees) Merr. BMC Plant Biol, 2018, 18:303.
doi: 10.1186/s12870-018-1525-9 pmid: 30477425
[22] 赵青山, 付颖, 叶非. 三唑并嘧啶磺酰胺类除草剂的研究概况. 植物保护, 2011, 37(2):14-19.
Zhao Q S, Fu Y, Ye F. Study summary of triazolo [1,5-a] pyrimidine-2-sulfonanilide herbicides. Plant Prot, 2011, 37(2):14-19 (in Chinese with English abstract).
[23] 刘伟堂. 小麦田牛繁缕(Myosoton aquaticum L. Moench.)对苯磺隆的抗性研究. 山东农业大学博士学位论文,山东泰安, 2015.
Liu W T. Study on the Resistance to Tribenuron-methyl in Water Chickweed (Myosoton aquaticum L. Moench.) in Wheat Fields. PhD Dissertation of Shandong Agricultural University, Tai’an, Shandong,China, 2015 (in Chinese with English abstract).
[24] Shaner D L, Anderson P C, Stidham M A. Imidazolinones: potent inhibitors of acetohydroxyacid synthase. Plant Physiol, 1984, 76:545-546.
doi: 10.1104/pp.76.2.545 pmid: 16663878
[25] Petit C, Duhieu B, Boucansaud K, Delye C. Complex genetic control of non-target-site-based resistance to herbicides inhibiting acetyl-coenzyme A carboxylase and acetolactate-synthase in Alopecurus myosuroides Huds. Plant Sci, 2010, 178:501-509.
doi: 10.1016/j.plantsci.2010.03.007
[26] Delye C, Pernin F, Scarabel L. Evolution and diversity of the mechanisms endowing resistance to herbicides inhibiting acetolactate-synthase (ALS) in corn poppy (Papaver rhoeas L.). Plant Sci, 2011, 180:333-342.
doi: 10.1016/j.plantsci.2010.10.007
[27] Scarabel L, Pernin F, Délye C. Occurrence, genetic control and evolution of non-target-site based resistance to herbicides inhibiting acetolactate synthase (ALS) in the dicot weedPapaver rhoeas. Plant Sci, 2015, 238:158-169.
doi: 10.1016/j.plantsci.2015.06.008 pmid: 26259184
[28] Yang Q, Deng W, Li X, Yu Q, Bai L, Zheng M. Target-site and non-target-site based resistance to the herbicide tribenuron- methyl in flixweed (Descurainia sophia L.). BMC Genomics, 2016, 17:551-563.
doi: 10.1186/s12864-016-2915-8
[29] Mei Y, Si C, Liu M, Qiu L, Zheng M. Investigation of resistance levels and mechanisms to nicosulfuron conferred by non-target- site mechanisms in large crabgrass (Digitaria sanguinalis L.) from China. Pestic Biochem Physiol, 2017, 141:84-89.
doi: S0048-3575(16)30210-3 pmid: 28911745
[30] Rey-Caballero J, Menéndez J, Osuna M D, Salas M, Torra J. Target-site and non-target-site resistance mechanisms to ALS inhibiting herbicides in Papaver rhoeas. Pestic Biochem Physiol, 2017, 138:57-65.
doi: S0048-3575(17)30082-2 pmid: 28456305
[31] Zhao B C, Fu D N, Yu Y, Huang C T, Yan K C, Li P S, Shafi J, Zhu H, Wei S H, Ji M S. Non-target-site resistance to ALS-inhibiting herbicides in a Sagittaria trifolia L. population. Pestic Biochem Physiol, 2017, 140:79-84.
doi: 10.1016/j.pestbp.2017.06.008
[32] Siminszky B, Corbin F T, Ward E R, Fleischmann T J, Dewey R E. Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides. Proc Natl Acad Sci USA, 1999, 96:1750-1755.
doi: 10.1073/pnas.96.4.1750
[33] Saika H, Horita J, Taguchi-Shiobara F, Nonaka S, Nishizawa-Yokoi A, Iwakami S, Hori K, Matsumoto T, Tanaka T, Itoh T, Yano M, Kaku K, Shimizu T, Toki S. A novel rice cytochrome P450 gene,CYP72A31, confers tolerance to acetolactate synthase-inhibiting herbicides in rice and Arabidopsis. Plant Physiol, 2014, 166:1232-1240.
doi: 10.1104/pp.113.231266
[1] SHI Lei, MIAO Li-Juan, HUANG Bing-Yan, GAO Wei, ZHANG Zong-Xin, QI Fei-Yan, LIU Juan, DONG Wen-Zhao, ZHANG Xin-You. Characterization of the promoter and 5'-UTR intron in AhFAD2-1 genes from peanut and their responses to cold stress [J]. Acta Agronomica Sinica, 2021, 47(9): 1703-1711.
[2] DAI Liang-Xiang, XU Yang, ZHANG Guan-Chu, SHI Xiao-Long, QIN Fei-Fei, DING Hong, ZHANG Zhi-Meng. Response of rhizosphere bacterial community diversity to salt stress in peanut [J]. Acta Agronomica Sinica, 2021, 47(8): 1581-1592.
[3] ZHANG Huan, LUO Huai-Yong, LI Wei-Tao, GUO Jian-Bin, CHEN Wei-Gang, ZHOU Xiao-Jing, HUANG Li, LIU Nian, YAN Li-Ying, LEI Yong, LIAO Bo-Shou, JIANG Hui-Fang. Genome-wide identification of peanut resistance genes and their response to Ralstonia solanacearum infection [J]. Acta Agronomica Sinica, 2021, 47(12): 2314-2323.
[4] LIU Shao-Rong, YANG Yang, TIAN Hong-Li, YI Hong-Mei, WANG Lu, KANG Ding-Ming, FANG Ya-Ming, REN Jie, JIANG Bin, GE Jian-Rong, CHENG Guang-Lei, WANG Feng-Ge. Genetic diversity analysis of silage corn varieties based on agronomic and quality traits and SSR markers [J]. Acta Agronomica Sinica, 2021, 47(12): 2362-2370.
[5] ZHANG Ping-Ping,YAO Jin-Bao,WANG Hua-Dun,SONG Gui-Cheng,JIANG Peng,ZHANG Peng,MA Hong-Xiang. Soft wheat quality traits in Jiangsu province and their relationship with cookie making quality [J]. Acta Agronomica Sinica, 2020, 46(4): 491-502.
[6] HUANG Bing-Yan,QI Fei-Yan,SUN Zi-Qi,MIAO Li-Juan,FANG Yuan-Jin,ZHENG Zheng,SHI Lei,ZHANG Zhong-Xin,LIU Hua,DONG Wen-Zhao,TANG Feng-Shou,ZHANG Xin-You. Improvement of oleic acid content in peanut (Arachis hypogaea L.) by marker assisted successive backcross and agronomic evaluation of derived lines [J]. Acta Agronomica Sinica, 2019, 45(4): 546-555.
[7] ZHAO Jia-Jia,MA Xiao-Fei,ZHENG Xing-Wei,HAO Jian-Yu,QIAO Ling,GE Chuan,WANG Ai-Ai,ZHANG Shu-Wei,ZHANG Xiao-Jun,JI Hu-Tai,ZHENG Jun. Effects of HMW-GS on wheat quality under different water conditions [J]. Acta Agronomica Sinica, 2019, 45(11): 1682-1690.
[8] HU Yi-Bo, YANG Xiu-Shi, LU Ping*,REN Gui-Xing*. Diversity and Correlation of Quality Traits in Quinoa Germplasms from North China [J]. Acta Agron Sin, 2017, 43(03): 464-470.
[9] CHEN Yu-Liang,SHI You-Tai,LUO Jun-Jie,LI Zhong-Wang,HOU Yi-Qing,WANG Di. Effect of Drought Stress on Agronomic Traits and Quality and WUE in Different Colored Upland Cotton Cultivars [J]. Acta Agron Sin, 2013, 39(11): 2074-2082.
[10] WANG Li-Hua, LI Jie-Qin, LIN Ping, WANG Pei, YOU Zhen-Wen, and ZHAN Qiu-Wen. Comparison of Effects of Sorghum bmr-6 and bmr-12 Genes on Yield and Quality Traits [J]. Acta Agron Sin, 2011, 37(07): 1308-1312.
[11] LI Ling, CHEN Jin-Gong, CHU Shui-Jin. Effects of Cadmium Stress on the Seed Quality Traits of Transgenic Cotton SGK3 and ZD-90 [J]. Acta Agron Sin, 2011, 37(05): 929-933.
[12] CHEN Mei-Xia, QI Jian-Min, WEI Cheng-Lin, XIE Zeng-Rong, LIN Pei-Qing, LAN Tao, TAO Ai-Fen, CHEN Tao. Preliminary Localization of Five Qualitative Traits in Kenaf Genetic Linkage Map [J]. Acta Agron Sin, 2011, 37(01): 165-169.
[13] TANG Yong-Lu, WU Yuan-Qi, ZHU Hua-Zhong, LI Chao-Su, LI Sheng-Rong, ZHENG Chuan-Gang, YUAN Ji-Chao, YU Xiu-Fang. Quality Performance and Stability of Main Wheat Cultivars in Sichuan Province [J]. Acta Agron Sin, 2010, 36(11): 1910-1920.
[14] WANG Ying, CHENG Li-Rui, LENG Jian-Tian, WU Cun-Xiang, HOU Wen-Sheng, HAN Tian-Fu. QTL Mapping of Agronomic and Quality Traits in Soybean under Different Post-Flowering Photoperiods [J]. Acta Agron Sin, 2010, 36(07): 1092-1099.
[15] SONG Dong-Ming,MA Dian-Rong,YANG Qing,CHEN Wen-Fu*. Effects of Weedy Rice on Yield and Quality and Micro-Ecological Environment in Cultivated Japonica Rice Population [J]. Acta Agron Sin, 2009, 35(5): 914-920.
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