银腺杨84K茎段瞬时转化体系的建立

doi:10.11707/j.1001-7488.20210409

摘要/Abstract

摘要：

目的: 我国需要大量人工林来满足木材需求，而速生林的木材品质有待改良。研究木材形成的基因调控是材性改良的基础。为加速鉴定木材形成相关基因的研究，建立一种快速、便捷、高效的瞬时转化体系尤为重要。方法: 采用显微切片，观察1年生银腺杨84K休眠茎段水培下的形成层活动规律；以其作为瞬时转化受体，增强型绿色荧光蛋白（eYGFP）作为报告基因，构建表达载体，采用农杆菌介导真空渗透侵染方法进行84K茎段的瞬时转化。并通过L₉（3⁴）正交试验对农杆菌GV3101侵染浓度、真空渗透侵染时间、真空渗透侵染次数及侵染后培养时间进行优化。结果: 银腺杨84K 1年生枝条室温水培条件下可以解除休眠，形成层在水培15天左右活动已比较旺盛，并出现新分化的导管。培养约30天，木质部大量积累。这说明枝条经1个月时间可以完成形成层的分化、木质部的形成等木材形成的全过程。从形态上观察，形成层、木质部和韧皮部的生长发育过程与正常树木没有差异。采用农杆菌介导真空渗透侵染方法，茎段及切片在激发光下可在形成层区域观察到绿色荧光，说明茎段转化成功。进一步通过正交试验分析，表明4个转化条件对转化率的影响依次为：侵染后水培天数>真空渗透侵染时间>菌液浓度>真空渗透侵染次数。农杆菌介导真空渗透瞬时转化银腺杨84K茎段的最优组合是农杆菌重悬液浓度OD₆₀₀为0.9、真空渗透侵染20 min、真空渗透侵染2次、侵染后水培15天。结论: 建立了农杆菌介导的银腺杨84K茎段真空渗透侵染瞬时转化体系。这一体系可快速鉴定参与维管组织分化相关基因的功能，有助于揭示木质部发育的调控机制。同时该方法也可为研究其他木本植物的木材形成相关基因提供借鉴。

关键词: 银腺杨84K, 茎段, 瞬时转化, 农杆菌介导, 真空渗透侵染, eYGFP瞬时表达

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

中图分类号:

S718.46

李晓军,安轶,黄李超,曾为,卢孟柱. 银腺杨84K茎段瞬时转化体系的建立[J]. 林业科学, 2021, 57(4): 82-89.

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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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

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