[1]张洁,郭文杰,蔡宣梅.东方百合高效转基因体系构建及EPSP基因的导入[J].北方园艺,2023,(04):46-53.[doi:10.11937/bfyy.20222378]
　ZHANG Jie,GUO Wenjie,CAI Xuanmei.Establishment of Efficient Transgenic System and Introduction of EPSP Gene Into Lilium[J].Northern Horticulture,2023,(04):46-53.[doi:10.11937/bfyy.20222378]
点击复制

东方百合高效转基因体系构建及EPSP基因的导入

《北方园艺》[ISSN:1001-0009/CN:23-1247/S] 卷: 期数: 2023年04 页码: 46-53 栏目: 出版日期: 2023-02-28
Title:
Establishment of Efficient Transgenic System and Introduction of EPSP Gene Into Lilium
作者:
张洁; 郭文杰; 蔡宣梅
（福建省农业科学院 生物技术研究所，福建 福州 350003）
Author(s):
ZHANG Jie; GUO Wenjie; CAI Xuanmei
(Biotechnology Institute，Fujian Academy of Agricultural Sciences，Fuzhou,Fujian 350003)
关键词:
百合; EPSP基因; 遗传转化; 转基因
Keywords:
Lilium; EPSP gene; transformation; transgene
DOI:
10.11937/bfyy.20222378
文献标志码:
A
摘要:
以东方百合‘西伯利亚’无菌鳞茎超薄切片（0.5~1.0 mm）为试材，通过PIC和2，4-D不同浓度筛选愈伤诱导培养条件，采用超薄鳞片直接分化及愈伤分化2种转化方式，测试对Cef、Kan及Hgy的耐受性，分析不同干燥预处理以及MES、AS对转化效果的影响，并通过EPSP基因的导入，验证基因转化阳性率及转化效果，以期优化百合遗传转化的技术参数，建立高效遗传转化体系并进行EPSP基因的导入。结果表明：PIC对百合愈伤诱导有良好效果，MS+1 mg?L-1 PIC+0.3 mg?L-1 NAA为最佳愈伤诱导培养基；抗生素敏感性测试发现，培养基添加75 mg?L-1 Kan或20 mg?L-1 Hyg结合250 mg?L-1 Cef适宜抗性筛选；超薄鳞片及愈伤在60 min和30 min的干燥预培养下，阳性率均显著提高；MS+10 mmol?L-1 MES+200 μmol?L-1 AS为重悬液处理，超薄鳞片获得48.3%的高效转化率，优于愈伤转化；同时，将EPSP基因转化百合，分子检测表示已获得转基因阳性株。该研究建立以超薄鳞片和愈伤为受体的2套高效遗传转化体系，并成功导入EPSP基因，获得800 mg?L-1的草甘膦抗性，为转基因技术在后续的生产应用奠定技术基础。
Abstract:
Ultrathin sections (0.5-1.0 mm) of aseptic bulbs of Lilium orientalis ‘Siberia’ were used as test materials.Callus induction culture conditions were screened by PIC and 2，4-D at different concentrations.The tolerance to Cef，Kan and Hgy was tested by direct section differentiation and callus differentiation.The effects of different drying pretreatment，MES and AS on the transformation effect were analyzed，and the positive rate of gene transformation and transformation effect were verified by the introduction of EPSP gene,in order to optimize the technical parameters of Lilium genetic transformation，establish an efficient genetic transformation system and introduce EPSP.The results showed that PIC had a good effect on callus induction.MS+1 mg?L-1 PIC+0.3 mg?L-1 NAA was the best medium for callus induction.Antibiotic sensitivity test showed that 75 mg?L-1 Kan or 20 mg?L-1 Hyg combined with 250 mg?L-1 Cef was suitable for resistance screening.The positive rates of ultrathin scales and callus were significantly increased after drying and preculture for 60 minutes and 30 minutes.MS+10 mmol?L-1 MES+200 μmol?L-1 AS treated with heavy suspension，the ultra-thin scales obtained an efficient conversion rate of 48.3%，which was better than callus transformation.At the same time，EPSP gene was transformed into Lilium，and molecular detection indicated that transgenic positive strains had been obtained.In this study，two sets of efficient genetic transformation systems of ultra-thin section and callus as the receptor were established，and EPSP gene was successfully introduced to obtain glyphosate resistance of 800 mg?L-1，laying a technical foundation for the subsequent production and application of transgenic technology.

参考文献/References:

[1]黄洁,刘晓华,管洁,等.百合分子育种研究进展[J].园艺学报，2012,39(9):1793-1808.[2]肖海燕,刘青林.百合等球根花卉育种研究进展[J].中国农业科技导报,2015,17(6)：21-28.[3]COHEN A，MEREDITH C P.Agrobacterium-mediated transformation of Lilium[J].Acta Horticulturae,1992,325:611-618.[4]MERCURI A，BENEDETTI L D，BRUNA S，et al.Agrobacterium-mediated transformation with rol genes of Lilium longiflorum Thumb[J].Acta Horticulturae，2003,612:129-136.[5]AZADI P，OTANG N V，CHIN D P,et al.Metabolic engineering of Lilium×formolongi using multiple genes of the carotenoid biosynthesis pathway[J].Plant Biotechnology Reports,2010(4):269-280.[6]YAN R，WANG Z，REN Y，et al.Establishment of efficient genetic transformation systems and application of CRISPR/Cas9 genome editing technology in Lilium pumilum DC.Fisch.and Lilium longiflorum White Heaven[J].Molecular Sciences，2019（20）:2920-2933.[7]HOSHI Y，KONDO M，MORI S，et al.Production of transgenic lily plants by Agrobacterium-mediated transformation[J].Plant Cell Reports，2004（22）:359-364.[8]WEI C，CUI Q，LIN Y，et al.Efficient Agrobacterium-mediated transformation of Lilium oriental ‘Sorbonne’ with genes encoding anthocyanin regulators[J].Canadian Journal of PlantScience,2017，97:796-807.[9]QI Y，DU L，QUAN Y,et al.Agrobacterium-mediated transformation of embryogenic cell suspension cultures and plant regeneration in Lilium tenuifolium oriental×trumpet ‘Robina’[J].Acta Physiologiae Plantarum,2014（36）:2047-2057.[10]王岚,刘雅莉,娄倩,等.超声波辅助农杆菌转化OT百合‘罗宾娜’的研究[J].西北农林科技大学学报（自然科学版）,2013，41(6):161-166.[11]CARLISLE S M，TREVORS J T.Glyphosate in the environment[J].Water Air and Soil Pollution,1988,39(3):409-420．[12]ALIBHAI M F,STALLINGS W C.Closing down on glyphosate inhibition with a new structure for drug discovery[J].Proceedings of the National Academy of Sciences of the United States of America,2001,98(6):2944-2946．[13]DU W S,WA11IS N G,MAZZULLA M J,et al.Characterization of streptococcus pneumoniae 5-enolpynevylshikimate-3-phosphate synthase and its actibation by univalent cations[J].European Journal of Biochemistry,2000,267:222-227.[14]GRASSOTTI A,GIMELLI F,GRASSOTTI A,et al.Bulb and cut flower production in the genus Lilium：Current status and the future[J].Acta Horticulturae,2011，900：21-36.[15]LAZARE S,BECHAR D,FERNIE A R,et al.The proof is in the bulb：Glycerol influences key stages of lily development[J].Plant Journal,2018，97，321-340.[16]田菲菲,刘雅莉,杜灵娟,等.农杆菌介导的ACO-RNAi载体对百合胚性愈伤的转化[J].西北农林科技大学学报（自然科学版）,2015,43(3):105-112.[17]张进忠,孙嘉曼,李朝生,等.百合AGPase基因RNAi载体构建及遗传转化研究[J].西南农业学报,2018,31(6):1097-1103.[18]SONG S,YAN R,WANG C，et al.Improvement of a genetic transformation system and preliminary study on the function of [STBX]LpABCB21 and LpPILS7[STBZ] based on somatic embryogenesis in Lilium pumilum DC.Fisch[J].Molecular Sciences,2020（21）:6784-6798.[19]张建鑫,刘雅莉,孟芮,等.农杆菌介导的百合ACO反义基因遗传转化的研究[J].西北农业学报,2008，17(6)：158-163.[20]尚爱芹,高永鹤,段龙飞,等.逆境诱导转录因子[STBX]AtDREB2A[STBZ]转化百合的研究[J].园艺学报，2014,41(1):149-156.[21]徐品三,刘岚,夏秀英.农杆菌介导的东方百合“西伯利亚”遗传转化体系建立[J].大连理工大学学报,2008,48(5):641-645.[22]赵欢蕊,刘雅莉,王跃进.农杆菌介导的普利安娜百合鳞茎直接分化受体系统的建立[J].西北农业学报,2007,16(4):142-147.[23]唐东芹,钱虹妹,黄丹枫,等.百合基因转化胚性愈伤组织受体系统的建立[J].浙江林学院学报,2003,20(3):273-276．[24]奚亚军,高海战,吕晓依,等.黄淮麦区推广品种小偃22农杆菌遗传转化体系的建立[J].核农学报，2009，23(2)：185-192.[25]URUSHIBARA S,TOZAWA Y,KAWAGISHI-KOBAYASHI M,et al.Efficient transformation of suspension-cultured rice cells mediated by Agrobacterium tumefaciens[J].Breeding Science，2001，51：33-38.[26]ARENCIBIA A D，CARMONA E R，TLLEZ P，et al.An efficient protocol for sugarcane (Saccharum spp.L.) transformation mediated by agrobacterium tumefaciens[J].Transgenic Research，1998（7）：213-222.[27]STACHEL S E,MESSEN E,van MONTAGU M,et al.Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens[J].Nature，1985，318:624-629.[28]邱金梅,温世杰,刘海燕,等.根癌农杆菌介导花生高效遗传转化体系的优化[J].中国油料作物学报,2010,32(2):208-211.[29]乔利仙,于新玲,隋炯明,等.农杆菌介导的花生遗传转化体系的优化[J].核农学报,2012,26(9):1244-1248.

相似文献/References:

[1]朱莉,李云飞,王忠义,等.秸秆发酵技术在百合生产中的应用[J].北方园艺,2013,37(04):74.
[2]王丽媛,周厚高.不同营养组合和施肥量对切花百合光合特性及干物质含量的影响[J].北方园艺,2014,38(04):58.
　WANG Li-yuan,ZHOU Hou-gao.Effect of Nutrient Combinations and Fertilizing Amount on the Photosynthetic Characteristics and Dry Matter of Cut-flower Lily[J].Northern Horticulture,2014,38(04):58.
[3]闫京艳,唐道城,巨秀婷,等.焦锑酸钾沉淀法在百合花柱钙分布研究中的应用[J].北方园艺,2014,38(01):78.
　YAN Jing-yan,TANG Dao-cheng,JU Xiu-ting,et al.Application of Potassium Pyroantimonate Precipitation on Calcium Distribution in the Style of Lily[J].Northern Horticulture,2014,38(04):78.
[4]赵兴华,吴海红,杨佳明.百合远缘杂交未成熟子房的离体培养研究[J].北方园艺,2014,38(04):90.
　ZHAO Xing-hua,WU Hai-hong,YANG Jia-ming.Study on Lily Distant Hybridization in vitroCulture of Immature Ovary[J].Northern Horticulture,2014,38(04):90.
[5]吴 超,丁晓瑜,黎 侠,等.东方百合“Siberia”基因组DNA提取方法比较研究[J].北方园艺,2014,38(02):108.
　WU Chao,DING Xiao-yu,LI Xia,et al.Comparative Study on Genomic DNA Extraction Methods From the Oriental Lily ‘Siberia’[J].Northern Horticulture,2014,38(04):108.
[6]周春华,尤超,陈凝华.百合组织培养研究进展[J].北方园艺,2013,37(14):193.
　ZHOU Chun-hua,YOU Chao,CHEN Ning-hua.Research Advances on Tissue Culture of Lilium brownie[J].Northern Horticulture,2013,37(04):193.
[7]夏九成,郑毅,吕会,等.利用百合花器官诱导试管苗快繁技术研究[J].北方园艺,2013,37(01):102.
　XIA Jiu-cheng,ZHENG Yi,LV Hui,et al.The Rapid Propagation of Lily by Inducing Seedling in Test Tube with Floral Organ[J].Northern Horticulture,2013,37(04):102.
[8]时婷婷,张桂玲,温四民,等.百合花粉超低温保存方法研究[J].北方园艺,2013,37(03):64.
　SHI Ting-ting,ZHANG Gui-ling,WEN Si-min,et al.Study on Crypreservation Methods of Lilium spp Pollens[J].Northern Horticulture,2013,37(04):64.
[9]黄海涛,王丹,陈霞.百合耐盐突变体同工酶电泳分析[J].北方园艺,2013,37(03):127.
　HUANG Hai-tao,WANG Dan,CHEN Xia.Isozyme Electrophoresis Analysis of Salt-tolerant Mutant of Lily[J].Northern Horticulture,2013,37(04):127.
[10]王建宇.外施赤霉素对休眠百合生理指标的影响[J].北方园艺,2012,36(02):64.
　WANG Jian-yu.Research on Effects of External GA3on Physiological Index of Dormancy of Lilium siberia[J].Northern Horticulture,2012,36(04):64.

备注/Memo

第一作者简介：张洁（1983-），女，硕士，助理研究员，现主要从事园艺作物生理及分子生物学等研究工作。E-mail：39508750@qq.com.基金项目：福建省属公益资助项目（2021R1027006）；福建省农业科学院自由探索科技创新资助项目（ZYTS2021006）；福建省农业科学院科技创新团队资助项目（CXTD2021009-3）。收稿日期：2022-06-09

更新日期/Last Update: 2023-03-21