发根农杆菌介导的尾巨桉遗传转化体系的建立

罗萍; 张昊楠; 徐建民; 胡冰; 王晓萍; 李光友; 范春节

发根农杆菌介导的尾巨桉遗传转化体系的建立

罗萍^1,2,
张昊楠^1,3,
徐建民¹,
胡冰¹,
王晓萍¹,
李光友¹,
范春节^1, ,

1. 林木遗传育种国家重点实验室国家林业和草原局热带林木培育重点实验室中国林业科学研究院热带林业研究所;
2. 南京林业大学;
3. 林木遗传育种国家重点实验室东北林业大学

基金项目:

中国林业科学研究院基本科研业务费专项资金项目(CAFYBB2020ZB004)

广东省基础与应用基础研究基金项目(2020A1515011059)~~

详细信息

作者简介:
罗萍(1997—),女,硕士研究生,主要从事林木遗传育种的研究

通讯作者:
范春节 E-mail:fanchunjie@caf.ac.cn

中图分类号: S792.39
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出版历程
- 收稿日期: 2021-12-02
- 刊出日期: 2022-05-19

Establishment of Agrobacterium rhizogenes-mediated Genetic Transformation System of Eucalyptus urophylla × E.grandis

1. State Key Laboratory of Tree Genetics and Breeding,Key Laboratory of State Forestry and Grassland Administration on Tropical Forestry,Research Institute of Tropical Forestry,Chinese Academy of Forestry;
2. Nanjing Forestry University;
3. State Key Laboratory of Tree Genetics and Breeding,Northeast Forestry University

摘要

摘要: 发根农杆菌（Agrobacterium rhizogenes）的建立对植物功能基因的验证具有重要意义，为了在桉树（Eucalyptus）中建立发根农杆菌介导的遗传转化体系，本研究以不同的发根农杆菌菌株侵染尾巨桉（Eucalyptus urophylla×E.grandis）的叶片和茎段，确定合适的农杆菌菌株和外植体类型，在此基础上开展农杆菌浓度、侵染时间对毛状根诱导的影响。结果表明：采用发根农杆菌菌株MSU440，以叶片为外植体进行发根诱导，最高获得了81.0%的毛状根诱导率，毛状根平均根长达到3.23 cm。在发根农杆菌浓度为OD600=0.3、侵染时间为30 min时，共培养48 h后经过20 mg·L^-1卡那霉素筛选培养，通过PCR分子鉴定和GUS染色证实外源基因稳定地整合在桉树毛状根基因组中，转化率达20.2%。初步建立了发根农杆菌介导的桉树遗传转化体系，为桉树基因功能鉴定和进一步的转基因育种奠定基础。
- 桉树 /
- 发根农杆菌 /
- 毛状根 /
- 转基因 /
- 转化
Abstract: Agrobacterium rhizogenes-mediated genetic transformation system plays an important role in the identification of functional genes.To establish Agrobacterium rhizogenes-mediated genetic transformation system in Eucalyptus,the different strains infected the leaves and stems of Eucalyptus urophylla × E.grandis were used to select the appropriate A.rhizogenes strains and explant types,and the effects of Agrobacterium concentration and infection time on hairy root induction were explored.The results showed that MSU440 was the optimal A.rhizogenes strain and leaves was the preferred explants for hairy root induction.The highest hairy root induction rate reached 81.0% with the average root length of hairy roots was 3.23 cm.The leaves were infected by MSU440 which grown to a bacterial density of 0.3（OD600）for 30 min,then they were co-cultured for 48h and transferred the selection medium supplemented with 20 mg·L^-1Kanamycin.The exogenous genes were stably integrated in the genome of Eucalyptus hair roots which confirmed by PCR analyses and GUS staining,and the transformation efficiency reached 20.2%.In our study,an A.rhizogenes-mediated genetic transformation system of Eucalyptus was preliminarily established,which laid a foundation for gene function identification and further transgenic breeding of Eucalyptus.
- Eucalyptus /
- Agrobacterium rhizogenes /
- hairy root /
- transgene /
- transformation

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参考文献(23)

[1]	谢耀坚.中国桉树育种研究进展及宏观策略[J].世界林业研究,2011,24(4):50-54. XIE Y J.Research progress on Eucalyptus breeding and its strategy in China[J].World Forestry Research,2011,24(4):50-54.
[2]	SONODA T,KOITA H,NAKAMOTO-OHTA S,et al.Increasing fiber length and growth in transgenic tobacco plants overexpressing a gene encoding the Eucalyptus camaldulensis HD-Zip classⅡtranscription factor[J].Plant Biotechnology,2009,26(1):115-120.
[3]	SPOKEVICIUS A V,SOUTHERTON S G,MACMILLANC P,et al.Beta-tubulin affects cellulose microfibril orientation in plant secondary fibre cell walls[J].The Plant Journal,2007,51(4):717-726.
[4]	邵志芳,陈伟元,罗焕亮,等.柞蚕抗菌肽D基因转化桉树培育抗青枯病株系的研究[J].林业科学,2002,38(2):92-97,178. SHAO Z F,CHEN W Y,LUO H L,et al.Studies on the introduction of the cecropin D gene into Eucalyptus urophylla to breed the resistant varieties to Pseudomonas solanacearum[J].Scientia Silvae Sinicae,2002,38(2):92-97,178.
[5]	VINING K J,ROMANEL E,JONES R C,et al.The floral transcriptome of Eucalyptus grandis[J].New Phytologist,2015,206(4):1406-1422.
[6]	AGGARWAL D,KUMAR A,REDDY M S.Genetic transformation of endo-1,4-β-glucanase(Korrigan)for cellulose enhancement in Eucalyptus tereticornis[J].Plant Cell,Tissue and Organ Culture:PCTOC,2015,122(2):363-371.
[7]	OUYANG L J,LI L M.Effects of an inducible aii A gene on disease resistance in Eucalyptus urophylla×Eucalyptus grandis[J].Transgenic Research,2016,25(4):441-452.
[8]	PLASENCIA A,SOLER M,DUPAS A,et al.Eucalyptus hairy roots,a fast,efficient and versatile tool to explore function and expression of genes involved in wood formation[J].Plant Biotechnology Journal,2016,14(6):1381-1393.
[9]	MENG D,YANG Q,DONG B Y,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.
[10]	BORTESI L,FISCHER R.The CRISPR/Cas9 system for plant genome editing and beyond[J].Biotechnology Advances,2015,33(1):41-52.
[11]	CAI Y,CHEN L,LIU X,et al.CRISPR/Cas9-mediated genome editing in soybean hairy roots[J].PLo S One,2015,10(8):e136064.
[12]	CUI M,LIU C,PIAO C,et al.A stable Agrobacterium rhizogenes-mediated transformation of cotton(Gossypium hirsutum L.)and plant regeneration from transformed hairy root via embryogenesis[J].Frontiers in Plant Science,2020,11:604255.
[13]	CRANE C,WRIGHT E,DIXON R A,et al.Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens-transformed roots and Agrobacterium rhizogenes-transformed hairy roots[J].Planta,2006,223(6):1344-1354.
[14]	DAI Y,HU G,DUPAS A,et al.Implementing the CRIS-PR/Cas9 technology in Eucalyptus hairy roots using wood-related genes[J].International Journal of Molecular Sciences,2020,21(10):3408.
[15]	肖璇.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].武汉:华中农业大学,2014. XIAO X.Development of Agrobacterium rhizogenes mediated transformation and canker disease resistance analysis in Citrus[D].Wuhan:Huazhong Agricultural University,2014.
[16]	GHARARI Z,BAGHERI K,DANAFAR H,et al.Enhanced flavonoid production in hairy root cultures of Scutellaria bornmuelleri by elicitor induced over-expression of MYB7 and FNSП2 genes[J].Plant Physiology and Biochemistry,2020,148:35-44.
[17]	刘雪羽,杜笑雪,陈思源,等.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[J].农业生物技术学报,2021,29(3):495-505. LIU X Y,DU X X,CHEN S Y,et al.Agrobacterium rhizogenes mediated high frequency hairy root induction system and genetic transformation in Betula luminifera[J].Journal of Agricultural Biotechnology,2021,29(3):495-505.
[18]	赖家业,石海明,刘凯,等.尾巨桉遗传转化系统建立的研究[J].四川大学学报(自然科学版),2007,44(2):415-419. LAI J Y,SHI H M,LIU K,et al.Studies on the establish genetic transformat of Eucalyptus urophylla×E.grandis[J].Journal of Sichuan University:Natural Science Edition,2007,44(2):415-419.
[19]	刘思巧.发根农杆菌介导的银杏毛状根培养及黄酮的生物合成[D].雅安:四川农业大学,2019.LIU S Q.Agrobacterium rhizogenes-mediated hairy root culture and flavonoid biosynthesis of Ginkgo biloba L.[D].Yaan:Sichuan Agricultural University,2019.
[20]	丁雪.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].四平:吉林师范大学,2017. DING X.Determination of physiological and optimization on the stem of Populus alba×P.berolinensis by Agrobacterium rhizogenes[D].Siping:Jilin Normal University,2017.
[21]	周利建,赵艳玲.巨尾桉组培快繁与转基因再生体系的优化[J].广东农业科学,2012,39(22):51-54. ZHOU L J,ZHAO Y L.Optimization of Eucalyptus grandis×E.ophylla tissue culture and regeneration system on genetic transformation[J].Guangdong Agricultural Sciences,2012,39(22):51-54.
[22]	MULLINS K V,LLEWELLYN D J,HARTNEY V J,et al.Regeneration and transformation of Eucalyptus camaldulensis[J].Plant Cell Reports,1997,16(11):787-791.
[23]	MORALEJO M,ROCHANGE F,BOUDET A M,et al.Generation of transgenic Eucalyptus globulus plantlets through Agrobacterium tumefaciens mediated transformation[J].Australian Journal of Plant Physiology,1998,25(2):207-212.

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