发根农杆菌介导的细茎柱花草毛状根转化体系的建立

doi:10.11733/j.issn.1007-0435.2023.02.032

草地学报 ›› 2023, Vol. 31 ›› Issue (2): 581-586.DOI: 10.11733/j.issn.1007-0435.2023.02.032

• 技术研发 • 上一篇

发根农杆菌介导的细茎柱花草毛状根转化体系的建立

赵兴坤¹, 孙昊¹, 杨丽云¹, 罗丽娟¹, 刘攀道²

1. 海南大学热带作物学院, 海南海口 570228;
2. 中国热带农业科学院热带作物品种资源研究所, 农业部华南作物基因资源与种质创制重点实验室, 海南海口 571101

收稿日期:2022-11-09 修回日期:2022-12-18 发布日期:2023-02-28
通讯作者: 罗丽娟,E-mail:luoljd@126.com;刘攀道,E-mail:liupandao@foxmail.com
作者简介:赵兴坤(1997-),男,汉族,硕士研究生,主要从事热带牧草转基因技术研究,E-mail:1607758669@qq.com
基金资助:
海南省重点研发计划科技合作方向项目(ZDYF2020211)；海南省科协青年科技英才学术创新计划项目(No.QCXM201901)；海南省自然科学基金高层次人才项目(No.322RC771)；中国科协青年人才托举工程第五届项目(No.2019QNRC001)资助

Establishment of the Genetic Transformation System of Stylosanthes gracilis mediated with Agrobacterium rhizogenes

ZHAO Xing-kun¹, SUN Hao¹, YANG Li-yun¹, LUO Li-juan¹, LIU Pan-dao²

1. College of Tropical Crops, Hainan University, Haikou, Hainan Province 570228, China;
2. Tropical Crop Genetic Resources Institute, Chinese Academy of Tropical Agriculture Sciences/Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China, Ministry of Agriculture and Rural Affairs, Haikou, Hainan Province 571101, China

Received:2022-11-09 Revised:2022-12-18 Published:2023-02-28

摘要/Abstract

摘要： 细茎柱花草(Stylosanthes gracilis)是圭亚那柱花草(S.guianensis)的野生近缘种，具有茎秆直立、种子落粒性低、抗旱能力较强等特点。为开发一种高效的细茎柱花草毛状根转化体系，本研究以细茎柱花草子叶节为外植体及4个发根农杆菌为供试菌种，探索菌液浓度、侵染时间、共培养时间对毛状根转化效率的影响。结果表明MSU440为诱导毛状根发生的最优菌株，诱导率为88.3%。其中，该菌株诱导毛状根产生的最佳条件为菌液浓度OD600=0.3、侵染时间为15 min、共培养4 d，转化效率达34.72%。同时，获得的转基因材料通过聚合酶链式反应(PCR)和蛋白质免疫印迹(Western blot)分子鉴定方式，证实了外源基因已整合到细茎柱花草毛状根的基因组中。因此，本研究成功建立了一种细茎柱花草的毛状根转基因体系。

关键词: 细茎柱花草, 子叶节, 发根农杆菌, 转基因, 毛状根

Abstract: Stylosanthes gracilis is a wild relative of S. guianensis，which has some advantages such as stem upright,Low seed shattering and strong drought resistance. In order to develop an efficient transformation system of the hairy root of S. gracilis, this study selected the cotyledonary nodes of S. gracilis as the explants and four strains of Agrobacterium rhizogenes as the test ones to explore the effects of bacterial solution concentration,infection time and co-culture time on the hairy root transformation efficiency. The results showed that the MSU440 was the best strain to induce that hairy roots with an induction rate of 88.3%. Further,OD600 0.3 bacterial solution concentration,15 min infection time and 4 d co-culture time was determined to be the best conditions for the generation of hairy roots induced by that strain,at which the transformation efficiency reached 34.72%. At the same time,the integration of exogenous genes into the genome of the hairy roots of S. gracilis was verified by the both polymerase chain reaction (PCR) and Western Blot detections. As a result,this study already successfully established a hairy root transgenic system of S. gracilis.

Key words: Stylosanthes. gracilis, Cotyledonary node, Agrobacterium rhizogenes, Genetic transformation, Hairy roots

中图分类号:

Q943.2

赵兴坤, 孙昊, 杨丽云, 罗丽娟, 刘攀道. 发根农杆菌介导的细茎柱花草毛状根转化体系的建立[J]. 草地学报, 2023, 31(2): 581-586.

ZHAO Xing-kun, SUN Hao, YANG Li-yun, LUO Li-juan, LIU Pan-dao. Establishment of the Genetic Transformation System of Stylosanthes gracilis mediated with Agrobacterium rhizogenes[J]. Acta Agrestia Sinica, 2023, 31(2): 581-586.

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参考文献

[1] FERREURA J J D S,PEREZ A P F,LEWIS G P,et al. Characteristics of the fruits of Brazilian species of Stylosanthes Sw. (Leguminosae) and their taxonomic value[J]. International Journal of Plant Sciences,2021,182(2):133-150
[2] RAMÍREZ M G V,TORRES D D C,BARRANTES J A G,et al. Soil recovery of alluvial gold mine spoils in the Peruvian Amazon using Stylosanthes guianensis,a promising cover crop[J]. Land Degradation and Development,2021,32(18):5143-5153
[3] 唐燕琼,吴紫云,刘国道,等. 柱花草种质资源研究进展[J]. 植物学报,2009,44(6):752-762
[4] 严琳玲,张瑜,白昌军. 13份柱花草品系生产性能比较[J]. 草业科学,2018,35(4):867-875
[5] SUN L,LANG C,CHEN Z,et al. Superior aluminium (Al) tolerance of Stylosanthes is achieved mainly by malate synthesis through an Al-enhanced malic enzyme,SgME1[J]. The New Phytologist,2014,202(1):106-115
[6] LIU P,CAI Z,CHEN Z,et al. A root-associated purple acid phosphatase,SgPAP23,mediates extracellular phytate-P utilization in Stylosanthes guianensis[J]. Plant Cell Environment,2018,41(12):2821-2834
[7] 张靖雪,盛伟,李欣勇,等. 柱花草低落粒种质筛选及农艺性状综合评价[J]. 草地学报,2022,30(1):108-117
[8] CALLES T,SCHUITZE-KRAFT R. Re-establishment of Stylosanthes gracilis (Leguminosae) at species level[J]. Kew Bulletin,2010,65(2):233-240
[9] MARQUES A,MORAES L,DOS SANTOS M A,et al. Origin and parental genome characterization of the allotetraploid Stylosanthes scabra Vogel (Papilionoideae,Leguminosae),an important legume pasture crop[J]. Annals of Botany,2018,122(7):1143-1159
[10] SMITH E F,TOWNSEND C O. A plant-tumor of bacterial origin[J]. Science,1907,25(643):671-673
[11] 王天佐,张文浩. 发根农杆菌介导的花苜蓿毛状根转化体系的建立[J]. 草地学报,2020,28(1):268-272
[12] YAN L,ZHAN C,DING L,et al. Development of a real-time PCR assay for the detection of Cladosporium fulvum in tomato leaves[J]. Journal of Applied Microbiology,2008,104:1417-1424
[13] 王升升,段珍,张吉宇. 发根农杆菌介导的白花草木樨毛状根转化体系的建立[J]. 草地学报,2021,29(11):2591-2599
[14] FOTI C,PAVLI O I. High-efficiency Agrobacterium rhizogenes-mediated transgenic hairy root induction of lens culinaris[J]. Agronomy,2020,10(8):1170
[15] 詹玉洁,刘博文,张仟,等. 发根农杆菌介导白羽扇豆转基因过表达体系的建立[J]. 植物生理学报,2021,57(3):655-660
[16] 罗萍,张昊楠,徐建民,等. 发根农杆菌介导的尾巨桉遗传转化体系的建立[J]. 植物研究,2022,42(3):512-520
[17] 周玲艳,姜大刚,吴豪,等. 潮霉素和PPT对水稻愈伤组织筛选效果的比较[J]. 仲恺农业技术学院学报,2003(2):10-15
[18] 韩秀丽,王文良,田田,等. 大麦转化中潮霉素和草丁膦的筛选效果研究[J].山东农业大学学报(自然科学版),2013,44(4):525-532
[19] DONG M,YANG Q,DONG B,et al. Development of an efficient root transgenic system for pigeon pea and its application to other important economically plants[J]. Plant Biotechnology Journal,2019,17(9):1804-1813
[20] CHENG Y,WANG X,CAO L,et al. Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene functional and gene editing analysis in soybean[J]. Plant Methods,2021,17(1):73
[21] 张金莲,刘金华,包涵,等. 丛枝菌根真菌与胡萝卜毛状根双重培养体系研究[J]. 热带作物学报,2020,41(8):1535-1542
[22] SHI M,LIAO P,NILE S H,et al. Biotechnological exploration of transformed root culture for value-added products[J]. Trends in Biotechnology,2021,39(2):137-149

发根农杆菌介导的细茎柱花草毛状根转化体系的建立

Establishment of the Genetic Transformation System of Stylosanthes gracilis mediated with Agrobacterium rhizogenes

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