农杆菌介导的楸树遗传转化体系

doi:10.11707/j.1001-7488.20210820

Abstract

Abstract:

Objective: Embryogenic callus of Catalpa bungei was used to establish an effective genetic transformation system, in order to provide a basis for the genetic improvement of C. bungei in the future. Method: Agrobacterium-mediated genetic transformation was carried out with embryogenic callus as explants. The optimal genetic transformation conditions were obtained through orthogonal test, and then exogenous genes were transferred into C. bungei. Result: Selective pressure screening was performed in 1/2 MS medium with different gradient concentrations of kanamycin. The differentiation rate of embryogenic callus was 0.00%, and the survival rate was only 5.71% in 1/2 MS medium supplemented with 60 mg·L^-1 Kana. Therefore, 60 mg·L^-1 Kana was identified as the selective pressure for genetic transformation of C. bungei. The orthogonal design was carried out by L₁₈(3⁷) table in Agrobacterium-mediated genetic transformation experiments. The transient expression rate was obtained by GUS histochemical staining. The intuitive analysis of the orthogonal test and one-way ANOVA were performed to the data obtained from the experiment. The results showed that Agrobacterium-mediated transformation efficiency was the highest under the conditions of 2 days of pre-culture, EHA105 of Agrobacterium strain, OD₆₀₀ 0.7 of liquid concentration, 300 μmol·L^-1 of acetyleugenone(AS), 10 min of infection, and 5 days of co-culture time. And the two factors that have the biggest influence on the transformation efficiency are AS concentration and pre-culture time. A total of 32 resistant tissue groups were obtained by screening and culture of the infected embryogenic callus for 8 months. PCR detection was conducted on 15 resistant callus with high proliferation. The results showed that 86.67% resistant tissue groups had exogenous genes integrated into the genome of C. bungei. Endogenous hormone levels affect somatic embryo differentiation, cytokinin(CTK) and abscisic acid(ABA) promote somatic embryogenesis, and auxin(IAA) and gibberellin(GA) inhibit somatic embryogenesis. According to the determination of endogenous hormones, the levels of CTK and ABA in transgenic resistant tissue were significantly lower than those in the embryogenic callus of wild-type, while the levels of IAA and GA were significantly higher than those in the wild-type callus. Therefore, the level of endogenous hormones may be the reason for the poor somatic embryo differentiation ability of transgenic resistant tissue of C. bungei. Conclusion: Through Agrobacterium-mediated transformation of the embryogenic callus of C. bungei, PCR detection was conducted on 15 screened resistant callus, among which 13 resistant callus had integration of exogenous genes. The somatic embryo differentiation ability of transgenic resistant tissues is poor, and the change of endogenous hormone level may be the reason that the resistant callus is difficult to differentiate.

Key words: Agrobacterium-mediated, Catalpa bungei, genetic transformation, endogenous hormones

CLC Number:

S718.46

Yunxin Cen,Jia Liu,Faju Chen,Jingyuan Yang,Qiang Liu,Tao Wang,Hongwei Liang. Agrobacterium-Mediated Genetic Transformation System of Catalpa bungei[J]. Scientia Silvae Sinicae, 2021, 57(8): 195-204.

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References 0

	傅玉兰, 费鹏飞, 刘小云. 楸树组培初代培养技术. 林业科技开发, 2009, 23 (4): 88- 91. doi: 10.3969/j.issn.1000-8101.2009.04.023
	Fu Y L , Fei P F , Liu X Y . Technique of inducing in vitro culture of Catalpa bungei C. A. Mey. China Forestry Science and Technology, 2009, 23 (4): 88- 91. doi: 10.3969/j.issn.1000-8101.2009.04.023
	高晗, 陈发菊, 王毅敏, 等. 楸树胚性细胞悬浮系的建立和植株再生. 基因组学与应用生物学, 2018, 37 (2): 895- 899.
	Gao H , Chen F J , Wang Y M , et al. Establishment of embryogenic cell suspension culture and plant regeneration of Catalpa bungei. Genomics and Applied Biology, 2018, 37 (2): 895- 899.
	韩创举, 杨培华, 樊军锋, 等. 楸树组培技术研究. 西北林学院学报, 2006, 21 (1): 80- 81. doi: 10.3969/j.issn.1001-7461.2006.01.019
	Han C J , Yang P H , Fan J F , et al. Tissue culture techniques of Catalpa bungei. Journal of Northwest Forestry University, 2006, 21 (1): 80. doi: 10.3969/j.issn.1001-7461.2006.01.019
	郝贵霞, 朱祯, 朱之悌. 毛白杨遗传转化系统优化的研究. 植物学报, 1999, (9): 3- 5.
	Hao G X , Zhu Z , Zhu Z T . Study on optimization of Populus tomentosa genetic transformation system. Journal of Integrative Plant Biology, 1999, (9): 3- 5.
	江荣翠, 彭方仁, 谭鹏鹏, 等. 楸树体细胞胚胎发生的研究. 南京林业大学学报: 自然科学版, 2010, 34 (2): 15- 18. doi: 10.3969/j.issn.1000-2006.2010.02.004
	Jiang R C , Peng F R , Tan P P , et al. Study on somatic embryogenesis of Catalpa bungei. Journal of Nanjing Forestry University: Natural Sciences Edition, 2010, 34 (2): 15- 18. doi: 10.3969/j.issn.1000-2006.2010.02.004
	江荣翠, 彭方仁, 谭鹏鹏. 滇楸体胚发生及生理生化特性研究. 林业科技开发, 2014, 28 (1): 25- 29.
	Jiang R C , Peng F R , Tan P P . Somatic embryogenesis and the physiological and biochemical characteristics in Catalpa fargesii Bur. f. duclouxii (Dode) Gilmour. China Forestry Science and Technology, 2014, 28 (1): 25- 29.
	金玉佩, 刘佳, 纪若璇, 等. 楸树体胚发生过程中5种酶的活性变化研究. 热带作物学报, 2017, 38 (2): 252- 257. doi: 10.3969/j.issn.1000-2561.2017.02.011
	Jin Y P , Liu J , Ji R X , et al. Analysis on activity change of enzyme during somatic embryogenesis in Catalpa bungei C. A. Mey. Journal of Tropical Crops, 2017, 38 (2): 252- 257. doi: 10.3969/j.issn.1000-2561.2017.02.011
	李玲, 顾恒, 岳远征, 等. 木本植物瞬时转化体系的研究进展. 分子植物育种, 2020, 18 (23): 7784- 7794.
	Li L , Gu H , Yue Y Z , et al. Advances in transient transformation systems of woody plants. Molecular Plant Breeding, 2020, 18 (23): 7784- 7794.
	梁有旺, 杜旭华, 王顺财, 等. 楸树嫩枝扦插生根的主要影响因子分析. 植物资源与环境学报, 2008, 17 (4): 46- 50. doi: 10.3969/j.issn.1674-7895.2008.04.009
	Liang Y W , Du X H , Wang S C , et al. Analysis of main influence factors on rooting of twig cutting of Catalpa bungei. Journal of Plant Resources and Environment, 2008, 17 (4): 46- 50. doi: 10.3969/j.issn.1674-7895.2008.04.009
	马玲玲, 王鹏, 王淑安, 等. 取材时间和激素对'豫楸1号' 腋芽诱导的影响. 北方园艺, 2014, (13): 84- 87.
	Ma L L , Wang P , Wang S A , et al. Effect of sampling time and hormones on axillary buds induction of 'Yuqiu No. 1'. Northern Horticulture, 2014, (13): 84- 87.
	孟路, 刘勇, 贺国鑫, 等. 楸树优良品种'朝霞'增殖及生根培养的研究. 西北林学院学报, 2019, 34 (1): 119- 123, 156. doi: 10.3969/j.issn.1001-7461.2019.01.17
	Meng L , Liu Y , He G X , et al. Multiplication and rooting culture of Catalpa bungei 'Zhaoxia'. Journal of Northwest Forestry University, 2019, 34 (1): 119- 123, 156. doi: 10.3969/j.issn.1001-7461.2019.01.17
	王长兰, 陈发菊, 金玉佩, 等. 楸树的开花生物学及繁育系统研究. 基因组学与应用生物学, 2015, 34 (9): 1981- 1987.
	Wang C L , Chen F J , Jin Y P , et al. Flower phenology and breeding system of Catalpa bungei (Bignoniaceae). Genomics and Applied Biology, 2015, 34 (9): 1981- 1987.
	王克臣. 2008. 亚麻离体再生及早期体细胞胚胎发生机理的研究. 哈尔滨: 东北农业大学博士学位论文.
	Wang K C. 2008. Study on mechanism of in vitro regeneration and initial somatic embryogenesis in flax. Harbin: PhD thesis of Northeast Agricultural University. [in Chinese]
	王义, 李仙, 赵文君, 等. 刺五加体细胞胚胎发生过程中生理变化研究. 中国中药杂志, 2008, 33 (17): 2182- 2186. doi: 10.3321/j.issn:1001-5302.2008.17.032
	Wang Y , Li X , Zhao W J , et al. Study on physiological changes during somatic embryogenesis of Acanthopanax senticosus. China Journal of Chinese Materia Medica, 2008, 33 (17): 2182- 2186.
	杨燕. 2008. 楸树组织培养研究. 南京: 南京林业大学博士学位论文.
	Yang Y. 2008. Tissue culture techniques of Catalpa bungei. Nanjing: PhD thesis of Nanjing Forestry University. [in Chinese]
	于永明, 王军辉, 麻文俊, 等. 不同浓度Kana、潮霉素对楸树试管苗生长的影响. 生物技术通讯, 2014, 25 (6): 832- 836. doi: 10.3969/j.issn.1009-0002.2014.06.019
	Yu Y M , Wang J H , Ma W J , et al. Effect of the kanamycin and hygromycin on Catalpa bungei in vitro culture. Biotechnology Newsletter, 2014, 25 (6): 832- 836. doi: 10.3969/j.issn.1009-0002.2014.06.019
	查丽燕, 宋舒晴, 王越, 等. 根癌农杆菌介导的巨大口蘑遗传转化体系的构建. 菌物学报, 2020, 7 (4): 1- 8.
	Zha L Y , Song S Q , Wang Y , et al. Construction of Agrobacterium-mediated transformation system in Macrocybe gigantea. Mycosystema, 2020, 7 (4): 1- 8.
	Barbara W , Małgorzata D G . LEAFY COTYLEDON2-mediated control of the endogenous hormone content: implications for the induction of somatic embryogenesis in Arabidopsis. Plant Cell, 2015, 121 (1): 255- 258.
	Braybrook S A , Harada J J . LECs go crazy in embryo development. Trends in Plant Science, 2008, 13, 624- 630. doi: 10.1016/j.tplants.2008.09.008
	Cangahuala-Inocente G C , Silveira V , Caprestano C A , et al. Dynamics of physiological and biochemical changes during somatic embryogenesis of Acca sellowiana. In Vitro Cellular & Developmental Biology-Plant, 2014, 50 (2): 166- 175.
	Confalonieri M , Balestrazzi A , Bisoffi S . Genetic transformation of Populus nigra by Agrobacterium tumefaciens. Plant Cell Reports, 1994, 13 (5): 256- 261.
	Dai W H , Cheng Z M , Sargent W . Plant regeneration and Agrobacterium-mediated transformation of two elite aspen hybrid clones from in vitro leaf tissues. In Vitro Cellular & Developmental Biology-Plant, 2003, 39, 6- 11.
	Igielski R , Kępczyńska E . Gene expression and metabolite profiling of gibberellin biosynthesis during induction of somatic embryogenesis in Medicago truncatula Gaertn. PLoS ONE, 2017, 12 (7): e0182055. doi: 10.1371/journal.pone.0182055
	Jayanthi M , Susanthi B , Murali M N , et al. In vitro somatic embryogenesis and plantlet regeneration from immature male inflorescence of adult dura and tenera palms of Elaeis guineensis (Jacq.). SpringerPlus, 2015, 4, 256. doi: 10.1186/s40064-015-1025-4
	Jiménez V M . Involvement of plant hormones and plant growth regulators on in vitro somatic embryogenesis. Plant Growth Regulation, 2005, 47 (2/3): 91- 110.
	Mcgranahan G H , Leslie C A , Uratsu S L , et al. Agrobacterium-mediated transformation of walnut somatic embryos and regeneration of transgenic plants. Nature Biotechnology, 1988, 6 (7): 800- 804. doi: 10.1038/nbt0788-800
	Movahedi A , Zhang J X , Amirian R , et al. An efficient Agrobacterium-mediated transformation system for poplar. International Journal of Molecular Sciences, 2014, 15 (6): 10780- 10793. doi: 10.3390/ijms150610780
	Priti M , Igor K . Agrobacterium-mediated stable genetic transformation of Populus angustifolia and Populus balsamifera. Frontiers in Plant Science, 2016, 7, 296.
	Song C , Lu L , Guo Y , et al. Efficient Agrobacterium-mediated transformation of the commercial hybrid poplar Populus alba× Populus glandulosa Uyeki. International Journal of Molecular Sciences, 2019, 20 (10): 2594. doi: 10.3390/ijms20102594
	Wang X , Liu S Q , Ji J , et al. Optimization of genetic transfomation system in somatic embryos of soybean mediated by Agrobacterium tumefaciens. Soybean Science, 2004, 23 (3): 200- 204.
	Xu W T , Zhang M , Wang C , et al. Somatic embryo induction and Agrobacterium-mediated transformation of embryonic callus tissue in Phoebe bournei, an endangered woody species in Lauraceae. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 2020, 48 (2): 572- 587. doi: 10.15835/nbha48211946

因素和 Sum of factors	GUS瞬时表达率GUS transient expression rate
因素和 Sum of factors	预培养时间 Pre-culture duration	菌株 Strain	菌液浓度 Bacteria concentration	AS浓度 AS concentration	侵染时间 Infection duration	共培养时间 Co-culture duration
K₁(%)	2.999	3.025	3.065	2.779	3.213	2.928
K₂(%)	3.329	3.229	3.214	3.038	3.146	3.140
K₃(%)	2.916	2.991	2.966	3.427	2.885	3.177
R(%)	0.413	0.238	0.247	0.648	0.327	0.249

内源激素 Endogenous hormones	回归方程 Regression equation	相关系数 R²
CTK	y= 27.275 x-0.808 8	0.999
IAA	y= 235.35 x-3.531 5	0.998
GA	y= 283.43 x-16.367 0	0.998
ABA	y= 175.23 x-6.167 3	0.999

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Agrobacterium-Mediated Genetic Transformation System of Catalpa bungei

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编号 No.	预培养时间 Pre-culture duration/d	菌株 Strain	菌液浓度 Bacteria concentration (OD₆₀₀)	AS浓度 AS concentration/(μmol·L^-1)	侵染时间 Infection duration/min	共培养时间 Co-culture duration/d	GUS瞬时表达率 GUS transient expression rate(%)
1	1	EHA105	0.5	100	10	3	43.57
2	1	GV3101	0.7	200	20	4	59.00
3	1	LBA4404	0.9	300	30	5	47.57
4	2	EHA105	0.5	200	20	5	53.94
5	2	GV3101	0.7	300	30	3	61.08
6	2	LBA4404	0.9	100	10	4	53.98
7	3	EHA105	0.7	100	30	4	35.46
8	3	GV3101	0.9	200	10	5	48.41
9	3	LBA4404	0.5	300	20	3	50.09
10	1	EHA105	0.9	300	20	4	55.01
11	1	GV3101	0.5	100	30	5	48.33
12	1	LBA4404	0.7	200	10	3	46.38
13	2	EHA105	0.7	300	10	5	68.41
14	2	GV3101	0.9	100	20	3	45.54
15	2	LBA4404	0.5	200	30	4	50.00
16	3	EHA105	0.9	200	30	3	46.11
17	3	GV3101	0.5	300	10	4	60.53
18	3	LBA4404	0.7	100	20	5	51.04