玉米根系原生质体制备及瞬时转化体系的建立

doi:10.13560/j.cnki.biotech.bull.1985.2026-0349

摘要/Abstract

摘要：

目的建立玉米根系原生质体的高效制备与瞬时转化方法，为玉米根系特异功能基因的研究提供技术支撑。方法以玉米自交系Mo17幼苗根系为材料，通过Box-Behnken响应面法，分别设计五因素三水平、三因素三水平试验，筛选原生质体制备（纤维素酶浓度、离析酶浓度、甘露醇浓度、酶解时间、根龄）与瞬时转化（质粒用量、PEG4000浓度、转化时间）的最优条件，测定不同组合下原生质体产量、活细胞比例及转化效率，并通过激素响应分析、亚细胞定位、Western-blot和双荧光素酶报告系统验证体系有效性。结果玉米根系原生质体制备的最优条件为2%纤维素酶、0.6%离析酶、0.6 mol/L甘露醇、酶解6 h、根龄9 d，此条件下原生质体产量达2.17×10⁶ cells/mL，活细胞率94.33%；瞬时转化的最优条件为每100 μL原生质体加10 μg质粒、PEG4000浓度40%、转化25 min，转化效率最高达65.1%。qPCR结果表明在原生质体中激素对其信号基因的激活作用与其在源组织中趋势一致；结合亚细胞定位、Western-blot及双荧光素酶报告实验验证，表明该体系可成功实现外源基因表达、蛋白亚细胞定位及转录调控分析。结论建立了玉米根系原生质体制备与瞬时转化体系，为玉米根系特异基因的功能解析及信号通路研究提供了高效可靠的技术手段。

关键词: 玉米, 根系, 原生质体, 瞬时转化体系, Box-Behnken响应面法

Abstract:

Objective This study aims to establish an efficient protoplast isolation and transient transformation system for maize roots, thereby providing technical support for studying maize root-specific functional genes. Method With roots of maize inbred line Mo17 seedlings as explants, Box-Behnken response surface method was used to design five-factor three-level and three-factor three-level experiments to optimize the conditions of protoplast isolation (cellulase concentration, macerozyme concentration, mannitol concentration, enzymatic hydrolysis time, root age) and transient transformation (plasmid dosage, PEG4000 concentration, transformation time), respectively. The protoplast yield, viable cell ratio and transformation efficiency under different condition combinations were evaluated. Furthermore, the system was verified by analysis in response to hormone treatment, subcellular localization, Western-blot and dual-luciferase reporter assays. Result The optimal isolation conditions were 2% cellulase, 0.6% macerozyme, 0.6 mol/L mannitol, 6 h enzymatic hydrolysis and 9-day-old roots, with a protoplast yield of 2.17×10⁶ cells/mL and a viable cell rate of 94.33%. The optimal transformation conditions were 10 μg plasmid per 100 μL protoplasts, 40% PEG4000 and 25 min transformation, achieving a maximum transformation efficiency of 65.1%. qPCR results showed that the activation of hormone-related genes by exogenous hormone treatment in protoplasts was consistent with that in the source tissues. Subsequent verification experiments proved that this system was successfully used for exogenous gene expression, protein subcellular localization and transcriptional regulation analysis. Conclusion This study established an efficient and reliable protoplast isolation and transient transformation system for maize roots, which offers a valuable technical tool for functional studies on maize root-specific genes.

Key words: maize, root, protoplast, transient transformation system, Box-Behnken response surface method

傅竞也, 燕杰, 裴文争, 包福琨, 杨越, 王丽平, 王强. 玉米根系原生质体制备及瞬时转化体系的建立[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0349.

FU Jing-ye, YAN Jie, PEI Wen-zheng, BAO Fu-kun, YANG Yue, WANG Li-ping, WANG Qiang. Establishment of a Protocol for Isolation and Transient Transformation of Maize Root Protoplasts[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0349.

图/表 9

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试验编号 Test No.	编码水平 Coded levels					实际水平 Actual levels					产量Yield ±SEM （×10⁶ cells/mL）	活细胞比例 FDA±SEM （%）
试验编号 Test No.	A	B	C	D	E	A	B	C	D	E	产量Yield ±SEM （×10⁶ cells/mL）	活细胞比例 FDA±SEM （%）
1	0	-1	0	1	0	1.5	0.4	0.6	8	7	1.65±0.21	90.62±0.77
2	0	0	0	0	0	1.5	0.6	0.6	6	7	1.46±0.14	87.12±1.39
3	-1	0	-1	0	0	1.0	0.6	0.4	6	7	1.52±0.08	88.16±0.96
4	0	0	0	-1	-1	1.5	0.6	0.6	4	5	1.81±0.06	90.31±1.21
5	1	0	-1	0	0	2.0	0.6	0.4	6	7	1.17±0.12	83.5±1.34
6	0	0	1	-1	0	1.5	0.6	0.8	4	7	1.08±0.13	89.2±0.73
7	0	0	0	1	-1	1.5	0.6	0.6	8	5	1.72±0.17	92.55±0.46
8	0	1	-1	0	0	1.5	0.8	0.4	6	7	1.47±0.12	96.38±0.71
9	1	0	0	-1	0	2.0	0.6	0.6	4	7	1.71±0.18	93.54±0.81
10	0	0	0	1	1	1.5	0.6	0.6	8	9	1.9±0.05	87.67±1.19
11	1	-1	0	0	0	2.0	0.4	0.6	6	7	1.59±0.2	91.06±0.82
12	0	1	0	-1	0	1.5	0.8	0.6	4	7	1.36±0.12	90.05±1.03
13	-1	0	0	0	-1	1.0	0.6	0.6	6	5	1.6±0.1	90.47±2.22
14	0	0	0	-1	1	1.5	0.6	0.6	4	9	1.31±0.06	89.62±1
15	0	-1	0	0	-1	1.5	0.4	0.6	6	5	1.57±0.05	87.7±1.92
16	0	0	1	0	-1	1.5	0.6	0.8	6	5	0.96±0.07	91.37±0.47
17	0	1	0	1	0	1.5	0.8	0.6	8	7	1.19±0.1	92.15±0.46
18	0	1	0	0	-1	1.5	0.8	0.6	6	5	1.36±0.1	93.39±1.66
19	-1	1	0	0	0	1.0	0.8	0.6	6	7	0.96±0.21	89.12±0.64
20	0	1	1	0	0	1.5	0.8	0.8	6	7	0.77±0.2	90.77±0.86
21	-1	0	0	0	1	1.0	0.6	0.6	6	9	1.08±0.18	92.16±3.89
22	1	0	0	0	-1	2.0	0.6	0.6	6	5	1.37±0.07	91.08±1.16
23	0	0	-1	1	0	1.5	0.6	0.4	8	7	1.63±0.02	90.15±1.38
24	0	0	1	0	1	1.5	0.6	0.8	6	9	1.29±0.19	93.19±1.16
25	0	0	0	0	0	1.5	0.6	0.6	6	7	1.27±0.16	93.86±0.31
26	0	0	0	0	0	1.5	0.6	0.6	6	7	1.26±0.17	91.02±0.89
27	0	-1	0	-1	0	1.5	0.4	0.6	4	7	1.13±0.27	94.77±1.26
28	0	1	0	0	1	1.5	0.8	0.6	6	9	1.63±0.29	92.37±1.67
29	1	0	0	0	1	2.0	0.6	0.6	6	9	2.17±0.1	94.83±0.28
30	0	0	-1	0	1	1.5	0.6	0.4	6	9	1.26±0.38	90.84±0.83
31	0	-1	0	0	1	1.5	0.4	0.6	6	9	1.4±0.02	85.56±1.12
32	0	-1	-1	0	0	1.5	0.4	0.4	6	7	1.38±0.09	94±0.18
33	0	0	-1	-1	0	1.5	0.6	0.4	4	7	1.19±0.11	87.59±1.25
34	1	1	0	0	0	2.0	0.8	0.6	6	7	1.3±0.08	88.85±2.71
35	1	0	0	1	0	2.0	0.6	0.6	8	7	1.75±0.05	91.29±1.13
36	0	0	-1	0	-1	1.5	0.6	0.4	6	5	1.73±0.48	90.82±1.38
37	-1	-1	0	0	0	1.0	0.4	0.6	6	7	1.1±0.27	94.22±1.49
38	-1	0	0	1	0	1.0	0.6	0.6	8	7	1.38±0.06	91.92±1
39	-1	0	1	0	0	0.4	0.6	0.8	6	7	0.33±0.01	90.25±1.04
40	0	-1	1	0	0	1.5	0.4	0.8	6	7	0.93±0.14	93.37±1.48
41	0	0	1	1	0	1.5	0.6	0.8	8	7	1.23±0.04	92.91±1.16
42	0	0	0	0	0	1.5	0.6	0.6	6	7	1.28±0.06	93.05±0.71
43	-1	0	0	-1	0	1.0	0.6	0.6	4	7	0.9±0.14	93.6±0.3
44	0	0	0	0	0	1.5	0.6	0.6	6	7	1.27±0.19	91.82±0.34
45	1	0	1	0	0	2.0	0.6	0.8	6	7	1.48±0.09	82.49±0.97
46	0	0	0	0	0	1.5	0.6	0.6	6	7	1.26±0.19	92.96±0.75

试验编号 Test No.	编码水平 Coded levels					实际水平 Actual levels					产量Yield ±SEM （×10⁶ cells/mL）	活细胞比例 FDA±SEM （%）
试验编号 Test No.	A	B	C	D	E	A	B	C	D	E	产量Yield ±SEM （×10⁶ cells/mL）	活细胞比例 FDA±SEM （%）
1	0	-1	0	1	0	1.5	0.4	0.6	8	7	1.65±0.21	90.62±0.77
2	0	0	0	0	0	1.5	0.6	0.6	6	7	1.46±0.14	87.12±1.39
3	-1	0	-1	0	0	1.0	0.6	0.4	6	7	1.52±0.08	88.16±0.96
4	0	0	0	-1	-1	1.5	0.6	0.6	4	5	1.81±0.06	90.31±1.21
5	1	0	-1	0	0	2.0	0.6	0.4	6	7	1.17±0.12	83.5±1.34
6	0	0	1	-1	0	1.5	0.6	0.8	4	7	1.08±0.13	89.2±0.73
7	0	0	0	1	-1	1.5	0.6	0.6	8	5	1.72±0.17	92.55±0.46
8	0	1	-1	0	0	1.5	0.8	0.4	6	7	1.47±0.12	96.38±0.71
9	1	0	0	-1	0	2.0	0.6	0.6	4	7	1.71±0.18	93.54±0.81
10	0	0	0	1	1	1.5	0.6	0.6	8	9	1.9±0.05	87.67±1.19
11	1	-1	0	0	0	2.0	0.4	0.6	6	7	1.59±0.2	91.06±0.82
12	0	1	0	-1	0	1.5	0.8	0.6	4	7	1.36±0.12	90.05±1.03
13	-1	0	0	0	-1	1.0	0.6	0.6	6	5	1.6±0.1	90.47±2.22
14	0	0	0	-1	1	1.5	0.6	0.6	4	9	1.31±0.06	89.62±1
15	0	-1	0	0	-1	1.5	0.4	0.6	6	5	1.57±0.05	87.7±1.92
16	0	0	1	0	-1	1.5	0.6	0.8	6	5	0.96±0.07	91.37±0.47
17	0	1	0	1	0	1.5	0.8	0.6	8	7	1.19±0.1	92.15±0.46
18	0	1	0	0	-1	1.5	0.8	0.6	6	5	1.36±0.1	93.39±1.66
19	-1	1	0	0	0	1.0	0.8	0.6	6	7	0.96±0.21	89.12±0.64
20	0	1	1	0	0	1.5	0.8	0.8	6	7	0.77±0.2	90.77±0.86
21	-1	0	0	0	1	1.0	0.6	0.6	6	9	1.08±0.18	92.16±3.89
22	1	0	0	0	-1	2.0	0.6	0.6	6	5	1.37±0.07	91.08±1.16
23	0	0	-1	1	0	1.5	0.6	0.4	8	7	1.63±0.02	90.15±1.38
24	0	0	1	0	1	1.5	0.6	0.8	6	9	1.29±0.19	93.19±1.16
25	0	0	0	0	0	1.5	0.6	0.6	6	7	1.27±0.16	93.86±0.31
26	0	0	0	0	0	1.5	0.6	0.6	6	7	1.26±0.17	91.02±0.89
27	0	-1	0	-1	0	1.5	0.4	0.6	4	7	1.13±0.27	94.77±1.26
28	0	1	0	0	1	1.5	0.8	0.6	6	9	1.63±0.29	92.37±1.67
29	1	0	0	0	1	2.0	0.6	0.6	6	9	2.17±0.1	94.83±0.28
30	0	0	-1	0	1	1.5	0.6	0.4	6	9	1.26±0.38	90.84±0.83
31	0	-1	0	0	1	1.5	0.4	0.6	6	9	1.4±0.02	85.56±1.12
32	0	-1	-1	0	0	1.5	0.4	0.4	6	7	1.38±0.09	94±0.18
33	0	0	-1	-1	0	1.5	0.6	0.4	4	7	1.19±0.11	87.59±1.25
34	1	1	0	0	0	2.0	0.8	0.6	6	7	1.3±0.08	88.85±2.71
35	1	0	0	1	0	2.0	0.6	0.6	8	7	1.75±0.05	91.29±1.13
36	0	0	-1	0	-1	1.5	0.6	0.4	6	5	1.73±0.48	90.82±1.38
37	-1	-1	0	0	0	1.0	0.4	0.6	6	7	1.1±0.27	94.22±1.49
38	-1	0	0	1	0	1.0	0.6	0.6	8	7	1.38±0.06	91.92±1
39	-1	0	1	0	0	0.4	0.6	0.8	6	7	0.33±0.01	90.25±1.04
40	0	-1	1	0	0	1.5	0.4	0.8	6	7	0.93±0.14	93.37±1.48
41	0	0	1	1	0	1.5	0.6	0.8	8	7	1.23±0.04	92.91±1.16
42	0	0	0	0	0	1.5	0.6	0.6	6	7	1.28±0.06	93.05±0.71
43	-1	0	0	-1	0	1.0	0.6	0.6	4	7	0.9±0.14	93.6±0.3
44	0	0	0	0	0	1.5	0.6	0.6	6	7	1.27±0.19	91.82±0.34
45	1	0	1	0	0	2.0	0.6	0.8	6	7	1.48±0.09	82.49±0.97
46	0	0	0	0	0	1.5	0.6	0.6	6	7	1.26±0.19	92.96±0.75

方差来源 Source of variance	平方和 Sum of squares	自由度 degree of freedom	均方 Mean square	F-value	P-value	显著性 Significance
Model	4.41	20	0.220 3	17.85	<0.000 1	**
A-纤维素酶	0.683 5	1	0.683 5	55.38	<0.000 1	**
B-离析酶	0.031 5	1	0.031 5	2.55	0.122 7	ns
C-甘露醇	0.530 3	1	0.530 3	42.97	<0.000 1	**
D-酶解时间	0.240 1	1	0.240 1	19.45	0.000 2	**
E-根龄	0.000 4	1	0.000 4	0.0324	0.858 6	ns
AB	0.005 6	1	0.005 6	0.4558	0.505 8	ns
AC	0.341 8	1	0.341 8	27.69	<0.000 1	**
AD	0.048 4	1	0.048 4	3.92	0.058 8	ns
AE	0.435 6	1	0.435 6	35.3	<0.000 1	**
BC	0.015 6	1	0.015 6	1.27	0.271 2	ns
BD	0.119	1	0.119	9.64	0.004 7	**
BE	0.048 4	1	0.048 4	3.92	0.058 8	ns
CD	0.021	1	0.021	1.7	0.203 7	ns
CE	0.16	1	0.16	12.96	0.001 4	**
DE	0.115 6	1	0.115 6	9.37	0.005 2	**
A²	0.063	1	0.063	5.11	0.032 8	*
B²	0.017 4	1	0.017 4	1.41	0.246 4	ns
C²	0.162 5	1	0.162 5	13.17	0.001 3	**
D²	0.157 1	1	0.157 1	12.73	0.001 5	**
E²	0.484 4	1	0.484 4	39.25	<0.000 1	**
残差	0.308 5	25	0.012 3
失拟误差	0.277 5	20	0.013 9	2.24	0.189 5	ns
纯误差	0.031	5	0.006 2
总离差	4.71	45

方差来源 Source of variance	平方和 Sum of squares	自由度 degree of freedom	均方 Mean square	F-value	P-value	显著性 Significance
Model	4.41	20	0.220 3	17.85	<0.000 1	**
A-纤维素酶	0.683 5	1	0.683 5	55.38	<0.000 1	**
B-离析酶	0.031 5	1	0.031 5	2.55	0.122 7	ns
C-甘露醇	0.530 3	1	0.530 3	42.97	<0.000 1	**
D-酶解时间	0.240 1	1	0.240 1	19.45	0.000 2	**
E-根龄	0.000 4	1	0.000 4	0.0324	0.858 6	ns
AB	0.005 6	1	0.005 6	0.4558	0.505 8	ns
AC	0.341 8	1	0.341 8	27.69	<0.000 1	**
AD	0.048 4	1	0.048 4	3.92	0.058 8	ns
AE	0.435 6	1	0.435 6	35.3	<0.000 1	**
BC	0.015 6	1	0.015 6	1.27	0.271 2	ns
BD	0.119	1	0.119	9.64	0.004 7	**
BE	0.048 4	1	0.048 4	3.92	0.058 8	ns
CD	0.021	1	0.021	1.7	0.203 7	ns
CE	0.16	1	0.16	12.96	0.001 4	**
DE	0.115 6	1	0.115 6	9.37	0.005 2	**
A²	0.063	1	0.063	5.11	0.032 8	*
B²	0.017 4	1	0.017 4	1.41	0.246 4	ns
C²	0.162 5	1	0.162 5	13.17	0.001 3	**
D²	0.157 1	1	0.157 1	12.73	0.001 5	**
E²	0.484 4	1	0.484 4	39.25	<0.000 1	**
残差	0.308 5	25	0.012 3
失拟误差	0.277 5	20	0.013 9	2.24	0.189 5	ns
纯误差	0.031	5	0.006 2
总离差	4.71	45

方差来源 Source of variance	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean square	F-value	P-value	显著性 Significance
Model	1 416.352	9	157.372 4	105.682 9	1.22E-06	**
A-质粒原生质体配比	74.118 4	1	74.118 4	49.773 98	0.000 201	**
B-PEG浓度	469.367 8	1	469.367 8	315.202 5	4.44E-07	**
C-转化时间	276.491 9	1	276.491 9	185.677 3	2.7E-06	**
AB	14.825 57	1	14.825 57	9.956 065	0.016 033	*
AC	138.661 3	1	138.661 3	93.117 54	2.7E-05	**
BC	220.405	1	220.405	148.012 3	5.8E-06	**
A²	108.933 9	1	108.933 9	73.154 22	5.93E-05	**
B²	341.482 9	1	341.482 9	229.321 8	1.32E-06	**
C²	229.521	1	229.521	154.134 1	5.06E-06	**
残差	10.423 7	7	1.489 099
失拟误差	1.463 695	3	0.487 898	0.217 812	0.879 535	ns
纯误差	8.96	4	2.24
总离差	1 426.775	16