发根农杆菌介导的光皮桦毛状根高频诱导体系及遗传转化

doi:10.3969/j.issn.1674-7968.2021.03.009

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Abstract A high frequency induced transformation system of hairy roots by Agrobacterium rhizogenes, that is an effective method to verify the gene function. Here, a genetic transformation system of Betula luminifera by A. rhizogenes was established, and had primarily optimized the culture condition of genetic transformation hairy root. First, 7 A. rhizogenes were used to infect B. luminifera leaves, and ArQual was obtained with the highest hairy root induction rate (69%). The optimal system for hairy root induction of B. luminifera was as follows: Leaves were pre-cultured in 1/2 MS medium for 2 days, infected for 20 minutes, and co-cultivation with acetosyringone (the concentration of 400 μmol/L) and cefotaxime (400 mg/L). In addition, the effect of different foreign gene vectors on hairy root induction was not significant. Based on the above system, pCAMBIA13011 vector containing β-glucosidase gene (GUS) gene and pGWB5 vector containing green fluorescent protein (GFP) gene were electroporated into ArQual, and transgenic hairy roots of B. luminifera were successfully induced with a conversion rate of 36.4%. In order to expand the application range of above genetic transformation system, the hypocotyls of B. luminifera seedlings (short cycle culture) were transformed by soaking and the stem segment of B. luminifera (long period culture) were punctured. After 30 days, the hairy roots were detected by GUS staining, fluorescence microscopy and PCR, and the results showed that GUS and GFP genes were successfully transformed and expressed, illustrated the different expression vector could express in new hairy roots of B. luminifera. In general, a high-frequency induction system for hairy roots of B. luminifera was established, which could be used to quickly verify gene function and expression in woody plants.

Key words： Betula luminifera Agrobacterium rhizogenes Hairy root Genetic transformation

Received: 27 June 2020 Published: 01 March 2021

ZTFLH:

S792.15

Corresponding Authors: *huanghh@zafu.edu.cn

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	LIU Xue-Yu
	DU Xiao-Xue
	CHEN Si-Yuan
	HU Xian-Ge
	HUANG Hua-Hong
	TONG Zai-Kang

Cite this article:

LIU Xue-Yu,DU Xiao-Xue,CHEN Si-Yuan, et al. Agrobacterium rhizogenes Mediated High Frequency Hairy Root Induction System and Genetic Transformation in Betula luminifera[J]. 农业生物技术学报, 2021, 29(3): 495-505.

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http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2021.03.009 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2021/V29/I3/495

[1] 毕宁, 周聿, 周应书, 等. 2017. 光皮桦作为天麻菌材树种的优势分析[J]. 现代农业科技, 2(17): 87-88.
(Bi N, Zhou Y, Zhou Y S, et al.2017. Advantage analysis on Betula luminifera H winkl as fungus material of Gastrodia elata blume[J]. Modern Agricultural Science and Technology, 2(17): 87-88.)
[2] 蔡媛, 钟灿, 谢芳一, 等. 2018. 基于响应面法对丹参毛状根诱导条件的优化研究[J]. 湖南中医杂志, 34(11): 144-147.
(Cai Y, Zhong C, Xie F Y, et al.2018. Optimization of induction conditions of Salvia miltiorrhiza hairy root based on the response surface method[J]. Journal of Agricultural Biotechnology, 34(11): 144-147.)
[3] 陈昆松, 李方, 徐昌杰, 等. 2004. 改良CTAB法用于多年生植物组织基因组DNA的大量提取[J]. 遗传, 4(23): 529-531.
(Chen K S, Li F, Xu C J, et al.2004. An efficient macro-method of genomic DNA isolation from Actinidia chinensis leaves[J]. Heredity, 4(23): 529-531.)
[4] 丁雪. 2017. 发根农杆菌介导的银中杨茎段遗传转化体系的建立及生理生化指标的测定[D]. 硕士学位论文, 吉林师范大学, 导师: 徐洪伟, pp. 32-37.
(Ding X.2017. Determination of physiological and optimization on the stem of Populus alba × P.berolinensis by Agrobacterium rhizogenes[D]. Thesis for M.S., Jilin Normal University, Suppervisor: Xu H Y, pp. 32-37.)
[5] 韩晓玲, 步怀宇, 郝建国, 等. 2006. 农杆菌转化的小冠花发状根的诱导及其植株再生[J]. 生物工程学报, 1(18): 107-113.
(Han X L, Bu H Y, Hao J G, et al.Hairy root induction and plant regeneration of Crownvetch(Coronilla varia L.) transformed by Agrobacterium rhizogenes[J]. Chinese Journal of Biotechnology, 1(18): 107-113.)
[6] 江成. 2014. 光皮桦BlOFPs基因的克隆及其功能研究[D], 硕士学位论文, 浙江农林大学, 导师: 黄华宏, 林二培, pp. 47-56.
(Jiang C.2014. Isolation and functional analysis of BlOFPs genes in Betula luminifera[D]. Thesis for M.S., Zhejiang A&F University, Supervisor: Huang H H, Lin E P., pp. 47-56.)
[7] 李明. 2017. 通过大豆毛状根体系快速验证GmNAC08、GmNAC06和GmNAC15的基因功能[D]. 博士学位论文, 石河子大学, 导师: 齐军仓, 张辉, pp. 81.
(Li M.2017. Rapid function validation of GmNAC08, GmNAC06 and GmNAC15 by Soybean hairy root[D]. Thesis for Ph.D., Shihezi University, Supervisor: Qi J C, Zhang H., pp. 81.)
[8] 林丽, 范海延, 潘野, 等. 2007. 发根农杆菌Ri质粒及其在植物次生代谢物质生产中的应用[J]. 北方园艺, 1(11): 94-97.
(Lin L, Fan H Y, Pan Y, et al.2007. Studies on Agrobacterium rhizogenes and its application to plant secondary metabolites[J]. Northern Horticulture, 1(11): 94-97.)
[9] 刘连旺, 张永清, 李先恩. 2015. 药用植物毛状根研究进展[J]. 山东中医药大学学报, 39(3): 288-291.
( Liu L W, Zhang Y Q, Li X E.2015. Study on hairy roots of medicinal plant[J]. Journal of Shangdong University of TCM, 39(3): 288-291.)
[10] 刘思巧. 2019. 发根农杆菌介导的银杏毛状根培养及黄酮的生物合成[D]. 硕士学位论文, 四川农业大学, 导师: 马明东, pp. 25-42.
(Liu S Q.2019. Agrobacterium rhizogenes-mediated hairy root culture and flavonoid biosynthesis of Ginkgo biloba L[D]. Thesis for M.S., Sichuan Agricultural University, Supervisor: Ma M D, pp. 25-42.)
[11] 孙晓敏. 2012. 光皮桦组织快繁及转基因体系建立[D]. 硕士学位论文, 浙江农林大学, 导师: 童再康, pp. 17-35.
(Sun X M.2012. Study on tissue culture and genetic transformation of Betula luminifera[D]. Thesis for M.S., Zhejiang A&F University, Supervisor: Tong Z K, pp. 17-35.)
[12] 王平勇, 徐永阳, 赵光伟, 等. 2019. 发根农杆菌介导西瓜转基因过表达体系的建立[J]. 果树学报, 36(12): 1763-1770.
(Wang P Y, Xu Y Y, Zhao G W, et al.2019. Preliminary study on Agrobacterium rhizogenes-mediated gene overexpression system in watermelon[J]. Journal of Fruit Science, 36(12): 1763-1770.)
[13] 王素娟, 裴月湖. 2000. 桦木属植物化学成分的研究进展[J]. 沈阳药科大学学报, 23(5): 378-382.
(Wang S J, Pei Y H.2000. A review on the chemical constituents of Betula[J]. Journal of Shenyang Pharmaceutical University, 23(5): 378-382.)
[14] 王天佐, 张文浩. 2020. 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, 28(1): 268-272.
(Wang T Z, Zhang W H.2020. Agrobacterium rhizogenes-mediated transformation of hairy roots in Medicago ruthenica[J]. Acta Agrestis Sinca, 28(1): 268-272)
[15] 向倩倩, 杨佳瑶, 侯梓淇, 等. 2019. 三叶青毛状根的诱导及其液体培养体系的研究[J]. 林业科技, 44(4): 5-9.
(Xiang Q Q, Yang J Y, Hou Z Q, et al.2019. Induction of Tetrastigma hemsleyanum hairy root and its liquid culture system[J]. Scientia Silvae Sinicae, 44(4): 5-9.)
[16] 肖璇. 2014. 发根农杆菌介导的柑橘遗传转化体系建立及转基因柑橘溃疡病抗性分析[D]. 博士学位论文, 华中农业大学, 导师: 郭文武, pp. 23-57.
(Xiao X.2014. Developmentof Agrobacterium rhizogenes mediated transformation and canker disease resistance analysis in Citrus [D]. Thesis for Ph.D., Huazhong Agricultural University, Supervisor: Guo W W, pp. 23-57.)
[17] 徐悦, 曹英萍, 王玉, 等. 2019. 发根农杆菌介导的菠菜毛状根遗传转化体系的建立[J]. 植物学报, 54(4): 515-521.
(Xu Y, Cao Y P, Wang Y, et al.2019. Agrobacterium rhizogenes-mediated transformation system of Spinacia oleracea[J]. Chinese Bulletin of Botany, 54(4): 515-521.)
[18] 杨俊松. 2016. 桂西北光皮桦人工林生物生产力、养分特性和水源涵养功能研究[D]. 硕士学位论文, 贵州大学, 导师: 王德炉, pp. 19-39.
(Yang J S.2016. Study on the biological productivity, nutrient characteristics and water conservation function of Betula luminifera plantation in Northwest Guangxi[D]. Thesis for M.S., Guizhou University, Supervisor: Wang D L, pp. 19-40.)
[19] 姚庆收, 武玉永, 于敏, 等. 2012. 发根农杆菌介导的橡胶树遗传转化体系的建立[J]. 安徽农业科学, 40(28): 13729-13730+13789 (Yao Q S, Wu Y Y, Yu M, et al. 2012. Establishment of Agrobacterium rhizogenes mediated genetic transformation system of Hevea brasiliensis[J]. Anhui Agricultural Science, 40(28): 13729-13730+13789.)
[20] 张利军, 宋小锋, 朱畇昊, 等. 2019. 冬凌草毛状根体系的建立[J]. 农村经济与科技, 30(4): 19-20.
(Zhang L J, Song X F, Zhu Y H, et al.2019. Establishment of hairy root system of Rabdosia rubescens[J]. Rural Economy and Science and Technology, 30(4): 19-20.)
[21] 张伦, 张熙, 吴建军, 等. 1999. 银杏毛状根的诱导和培养[J]. 贵州科学, 2(17): 132-134.
(Zhang L, Zhang X, Wu J, et al.1999. Induction and culture of hairy roots of Ginkgo biloba[J]. Guizhou Science, 2(17): 132-134.)
[22] Balasubramanian A, Venkatachalam R, Selvakesavan K R, et al.2011. Optimisation of methods for Agrobacterium rhizogenes mediated generation of composite plants in Eucalyptus camaldulensis[J]. BMC Proceedings, 5(S7): 45.
[23] Boisson-Dernier A, Chabaud M, Garcia F, et al.2001. Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations[J]. Molecular Plant-Microbe Interactions, 14(6): 695-700.
[24] Bosselut N, van Ghelder C, Claverie M, et al.2011. Agrobacterium rhizogenes-mediated transformation of Prunus as an alternative for gene functional analysis in hairy-roots and composite plants[J]. Plant Cell Reports, 30(7): 1313-1326.
[25] Chandra S.2012. Natural plant genetic engineer Agrobacterium rhizogenes: Role of T-DNA in plant secondary metabolism[J]. Biotechnology Letters, 34(3): 407-415.
[26] Gangopadhyay M, Dewanjee S, Bhattacharyya S, et al.2010. Effect of different strains of Agrobacterium rhizogenes and nature of explants on Plumbago indica hairy root culture with special emphasis on root biomass and plumbagin production[J]. Natural Product Communications, 5(12): 1913-1916
[27] Godwin I, Todd G, Ford-Lloyd B, et al.1991. The effects of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species[J]. Plant Cell Reports, 9(12): 671-675.
[28] Han K H, Meilan R, Ma C, et al.2000. An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids (genus Populus)[J]. Plant Cell Reports, 19(3): 315-320.
[29] Howe G T, Goldfarb B, Strauss S H, et al.1994. Agrobacterium-mediated transformation of hybrid poplar suspension cultures and regeneration of transformed plants[J]. Plant cell, Tissue and Organ Culture, 36(01): 59-71.
[30] Meng D, Yang Q, Dong B, et al.2019. Development of an efficient root transgenic system for pigeon pea and its application to other important economically plants[J]. Plant Biotechnology Journal, 17(9): 1804-1813.
[31] Plasencia A, Soler M, Dupas A, et al.2016. Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation[J]. Plant Biotechnology Journal, 14(6): 1381-1393.
[32] Weller S A, Stead D E, Young J P W.2004. Acquisition of an Agrobacterium Ri plasmid and pathogenicity by other alpha-Proteobacteria in cucumber and tomato crops affected by root mat[J]. Applied and Environmental Microbiology, 70(5): 2779-2785.
[33] Zych M, Pietrosiuk A, Karasiewicz M, et al.2008. Establishment of Rhodiola kirilowii hairy roots using Agrobacterium rhizogenes LBA 9402[J]. Herba Polonica, 54(4): 7-16.