银腺杨84K茎段瞬时转化体系的建立

doi:10.11707/j.1001-7488.20210409

Abstract

Abstract:

Objective: A large scale of plantation are needed to meet the demand for timber in China and the wood quality of fast-growth plantations needs to be improved. The study of gene regulation of wood formation is the basis for improvement of wood quality. In order to reveal the molecular mechanisms on the wood development, it is particularly important to establish a fast, convenient and efficient transient transformation system. Method: Microsections were used to observe the cambium activity of the 1-year-old poplar (Populus alba×P. glandulosa '84K') dormancy-released stems under hydroponic culture as a transient transformation receptor. The enhanced green fluorescent protein (eYGFP) was used as a reporter gene to be constructed in an expression vector. The Agrobacterium-mediated vacuum infiltration method was used for transient transformations. The L₉(3⁴) orthogonal test was used to optimize the Agrobacterium GV3101 infiltration concentration, vacuum infiltration transformation time, vacuum infiltration transformation frequency and culture time after transformation. Result: The dormancy of annual shoots was released under water culture at room temperature. The cambium had been active in water for 15 days, and new vessels had appeared. Abundant xylem cells accumulated in about 30 days. It showed that the stems could go through the whole process of wood formation, such as the differentiation of cambium and the formation of xylem within one month. From the morphological, development processes of cambium, xylem and phloem were not different from those of normal trees. Using Agrobacterium-mediated vacuum infiltration method, green fluorescence observation under excitation light in the stem slice indicated the transformation result of the stem. Through orthogonal test analysis, the influence of the four conditions was ranked as follows: culture time > vacuum infiltration transformation time > Agrobacterium concentration > vacuum infiltration transformation frequency. The optimal combination is that the Agrobacterium concentration (OD₆₀₀) is 0.9, vacuum infiltration transformation time is 20 min, vacuum infiltration transformation frequency is 2, and culture 15 days after transformation. Conclusion: The Agrobacterium-mediated vacuum infiltration transient transformation system of poplar '84K' stem segments was established. This system can be used to identify the functions of genes involved in vascular tissue differentiation in a short time, which is helpful for the study of the regulation mechanism of xylem development. This method also provides a basis for the study of related genes in other woody plants.

Key words: Populus alba×P. glandulosa '84K', stem segment, transient transformation, Agrobacterium-mediated transformation, vacuum infiltration transformation, eYGFP transient expression

CLC Number:

S718.46

Xiaojun Li,Yi An,Lichao Huang,Wei Zeng,Mengzhu Lu. Establishment of A Transient Transformation System for Stem Segments of Poplar 84K[J]. Scientia Silvae Sinicae, 2021, 57(4): 82-89.

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References 0

	An Y , Zhou Y , Han X , et al. The GATA transcription factor GNC plays an important role in photosynthesis and growth in poplar. Journal of Experimental Botany, 2020, 71 (6): 1969- 1984. doi: 10.1093/jxb/erz564
	Chin D P , Shiratori I , Shimizu A , et al. Generation of brilliant green fluorescent petunia plants by using a new and potent fluorescent protein transgene. Scientific Reports, 2018, 8 (1): 16556. doi: 10.1038/s41598-018-34837-2
	de Oliveira M L , Febres V J , Costa M G , et al. High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration. Plant Cell Reports, 2009, 28 (3): 387- 395. doi: 10.1007/s00299-008-0646-2
	Fizree P M A A , Shaharuddin N A , Ho C L , et al. Evaluation of transient DsRED gene expression in oil palm embryogenic calli. Scientia Horticulturae, 2019, 257, 108679. doi: 10.1016/j.scienta.2019.108679
	Gambino G , Gribaudo I . Genetic transformation of fruit trees: current status and remaining challenges. Transgenic Research, 2012, 21 (6): 1163- 1181. doi: 10.1007/s11248-012-9602-6
	Guo Y , Song X , Zhao S , et al. A transient gene expression system in Populus euphratica Oliv. protoplasts prepared from suspension cultured cells. Acta Physiologiae Plantarum, 2015, 37 (8): 1- 8. doi: 10.1007/s11738-015-1906-8
	Han K H , Meilan R , Ma C , et al. An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids (genus Populus). Plant Cell Reports, 2000, 19 (3): 315- 320. doi: 10.1007/s002990050019
	Han Y , Wu M , Cao L , et al. Characterization of OfWRKY3, a transcription factor that positively regulates the carotenoid cleavage dioxygenase gene OfCCD4 in Osmanthus fragrans. Plant Molecular Biology, 2016, 91 (4/5): 485- 496.
	Huang X , Chen S , Peng X , et al. An improved draft genome sequence of hybrid Populus alba×Populus glandulosa. Journal of Forestry Research, 2020, 20, 1- 10. doi: 10.1007/s11676-020-01235-2
	Jansson S , Douglas C J . Populus: a model system for plant biology. Annual Review of Plant Biology, 2007, 58 (1): 435- 458. doi: 10.1146/annurev.arplant.58.032806.103956
	Koroleva O A , Tomlinson M L , Leader D , et al. High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions. The Plant Journal, 2005, 41 (1): 162- 174.
	Kumagai M H , Donson J , Della-Cioppa G , et al. Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proceedings of the National Academy of Sciences of the United States of America, 1995, 92 (5): 1679- 1683. doi: 10.1073/pnas.92.5.1679
	Lv Y D , Zhang M L , Wu T N , et al. The infiltration efficiency of Agrobacterium-mediated transient transformation in four apple cultivars. Scientia Horticulturae, 2019, 256, 108597. doi: 10.1016/j.scienta.2019.108597
	Ma J , Wan D , Duan B , et al. Genome sequence and genetic transformation of a widely distributed and cultivated poplar. Plant Biotechnology Journal, 2019, 17 (2): 451- 460. doi: 10.1111/pbi.12989
	Ma T , Wang J , Zhou G , et al. Genomic insights into salt adaptation in a desert poplar. Nature Communications, 2013, 4 (1): 241- 458.
	Mo R , Yang S , Zhang Q , et al. Vacuum infiltration enhances the Agrobacterium-mediated transient transformation for gene functional analysis in persimmon (Diospyros kaki Thunb.). Scientia Horticulturae, 2019, 251, 174- 180. doi: 10.1016/j.scienta.2019.03.002
	Nowak K , Luniak N , Meyer S , et al. Fluorescent proteins in poplar: a useful tool to study promoter function and protein localization. Plant Biology, 2004, 6 (1): 65- 73. doi: 10.1055/s-2004-815730
	Polle A , Janz D , Teichmann T , et al. Poplar genetic engineering: promoting desirable wood characteristics and pest resistance. Applied Microbiology and Biotechnology, 2013, 97 (13): 5669- 5679. doi: 10.1007/s00253-013-4940-8
	Simmons C W , Vander Gheynst J S , Upadhyaya S K . A model of Agrobacterium tumefaciens vacuum infiltration into harvested leaf tissue and subsequent in planta transgene transient expression. Biotechnology & Bioengineering, 2009, 102 (3): 965- 970.
	Spolaore S , Trainotti L , Casadoro G . A simple protocol for transient gene expression in ripe fleshy fruit mediated by Agrobacterium. Journal of Experimental Botany, 2001, 52 (357): 845- 850. doi: 10.1093/jexbot/52.357.845
	Takata N , Eriksson M E . A simple and efficient transient transformation for hybrid aspen (Populus tremula×P. tremuloides). Plant Methods, 2012, 8 (1): 30. doi: 10.1186/1746-4811-8-30
	Tan B , Xu M , Chen Y , et al. Transient expression for functional gene analysis using Populus protoplasts. Plant Cell Tissue & Organ Culture, 2013, 114 (1): 11- 18. doi: 10.1007%2Fs11240-013-0299-x
	Trivellini A , Ferrante A , Hunter D A , et al. Agrobacterium tumefaciens-mediated transformation of axillary bud callus of Hibiscus rosa-sinensis L. 'Ruby' and regeneration of transgenic plants. Plant Cell, Tissue and Organ Culture, 2015, 121 (3): 681- 692. doi: 10.1007/s11240-015-0738-y
	Tuskan G A , Difazio S , Jansson S , et al. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science, 2006, 313 (5793): 1596- 1604. doi: 10.1126/science.1128691
	Ueno K , Fukunaga Y , Arisumi K . Genetic transformation of Rhododendron by Agrobacterium tumefaciens. Plant Cell Reports, 1996, 16 (1/2): 38- 41. doi: 10.1007/BF01275445
	van Nocker S , Gardiner S E . Breeding better cultivars, faster: applications of new technologies for the rapid deployment of superior horticultural tree crops. Horticulture Research, 2014, 1, 14022. doi: 10.1038/hortres.2014.22
	Wu S L , Yang X B , Liu L Q , et al. Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves. Bioscience Biotechnology and Biochemistry, 2015, 79 (8): 1- 6.
	Yang L , Sun Y , Xie L , et al. A novel approach for in situ bud transformation of Populus by Agrobacterium. Scandinavian Journal of Forest Research, 2009, 25 (1): 3- 9.
	Yang Y , Li R , Qi M . In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant Journal, 2000, 22 (6): 543- 551. doi: 10.1046/j.1365-313x.2000.00760.x
	Yoo S D , Cho Y H , Sheen J . Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols, 2007, 2 (7): 1565- 1572. doi: 10.1038/nprot.2007.199
	Zheng L , Liu G , Meng X , et al. A versatile Agrobacterium-mediated transient gene expression system for herbaceous plants and trees. Biochemical Genetics, 2012, 50 (9/10): 761- 769. doi: 10.1007/s10528-012-9518-0

试验号 Test No.	因素 Factors				eYGFP瞬时转化率 eYGFP transient transformation rate (%)
试验号 Test No.	A：菌液浓度 Agrobacterium concentration (OD₆₀₀)	B：真空渗透侵染时间 Vacuum infiltration transformation time/min	C：真空渗透侵染次数 Vacuum infiltration transformation frequency	D：水培天数 Culture time/d	eYGFP瞬时转化率 eYGFP transient transformation rate (%)
1	0.6	15	2	9	6.33±3.17
2	0.6	20	3	12	22.40±2.97
3	0.6	25	4	15	18.23±0.77
4	0.9	15	4	12	21.30±2.44
5	0.9	20	2	15	29.90±1.72
6	0.9	25	3	9	7.43±1.83
7	1.2	15	3	15	17.63±0.47
8	1.2	20	4	9	7.67±2.85
9	1.2	25	2	12	21.73±2.78

方差来源 Sources of variance	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean square	F	P
A：菌液浓度 Agrobacterium concentration	0.009	2	0.005	2.834	0.085
B：真空渗透侵染时间 Vacuum infiltration transformation time	0.013	2	0.006	3.963	0.037^*
C：真空渗透侵染次数 Vacuum infiltration transformation frequency	0.008	2	0.004	2.369	0.122
D：水培天数 Culture time	0.130	2	0.065	40.845	0.000^*
误差 Error	0.029	18	0.002
总计 Total	0.964	27

Establishment of A Transient Transformation System for Stem Segments of Poplar 84K

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