优化子叶节转化法培育大豆MtDREB2A转基因植株

doi:10.11983/CBB16257

摘要/Abstract

摘要： 将正交因素试验与GUS基因组织化学染色等技术相结合, 优化大豆(Glycine max)品种东农50遗传转化体系, 导入抗旱关键基因MtDREB2A。结果表明, 大豆种子表面消毒, NaClO溶液法与Cl₂气熏蒸法的去污染率分别达到98.67%和93.33%。子叶节法转GUS基因组织化学染色率(68.33%)显著高于下胚轴法(14.00%)和胚尖法(0.67%) (P<0.05)。种子萌发5天, 农杆菌(Agrobacterium tumefaciens)培养温度25°C, OD₆₀₀=0.9, 共培养5天的转GUS基因子叶节最高达72.00%; 恢复培养5天, 草丁膦(3 mg·L^-1)、头孢噻肟钠(200 mg·L^-1)和羧苄青霉素(300 mg·L^-1)筛选诱导分化的转GUS基因不定芽最多为3.33%; 优化的大豆遗传转化体系转化效率为1.11%。转MtDREB2A基因大豆东农50植株根系更加密集, 主根长度和侧根数量均显著高于对照(P<0.05), 证实MtDREB2A基因具有促进大豆根系生长的作用, 为利用该基因进行大豆抗旱育种奠定了坚实的基础并提供了理论依据。

关键词: 大豆, 遗传转化体系优化, GUS基因, MtDREB2A基因, 根系改良

Abstract: Orthogonal factorial experiments and histochemical GUS staining were combined to optimize the genetic transformation system of Glycine max cv. ‘Dongnong 50’ and transfer the key gene MtDREB2A for drought resistance into the soybean. Sterile of soybean seeds used as explants with NaClO solution and Cl₂ gas fumigation methods reached 98.67% and 93.33% germination, respectively. Histochemical staining rate of the tissues transformed with GUS by the cotyledon node method was 68.33%, significantly higher than that by the hypocotyl (14.00%) and embryo tip (0.67%) methods (P<0.05). The cotyledon node-transformed GUS gene was up to 72.00% in germinated sterile seeds for 5 days, mediated by Agrobacterium tumefaciens cultured at 25°C, OD₆₀₀ 0.9, and co-cultured for 5 days. The shoots were induced and differentiated with cotyledon node-transformed GUS up to 3.33% by optimal recovery culture for 5 days and were screened on culture medium containing phosphinothricin (3 mg·L^-1), cefotaxime sodium (200 mg·L^-1) and carbenicillin (300 mg·L^-1). The transgenic efficiency was 1.11% with the optimized soybean genetic transformation system. The MtDREB2A transgenic plant roots of soybean ‘Dongnong 50’ were more dense and both taproot length and lateral root number were significantly longer and greater than those of the control (P<0.05). The study verified that the MtDREB2A gene plays a role in promoting soybean root growth, which lays a solid foundation and provides a theoretical basis for the gene using in drought resistance breeding of soybean.

Key words: soybean, optimization of gene transformation system, GUS, MtDREB2A, root enhancement

吴国栋, 修宇, 王华芳. 优化子叶节转化法培育大豆MtDREB2A转基因植株. 植物学报, 2018, 53(1): 59-71.

Guodong Wu, Yu Xiu, Huafang Wang. Breeding of MtDREB2A Transgenic Soybean by an Optimized Cotyledonary-Node Method. Chinese Bulletin of Botany, 2018, 53(1): 59-71.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB16257

https://www.chinbullbotany.com/CN/Y2018/V53/I1/59

图/表 10

表1 植物表达载体构建所需引物

Table 1 The primers used in construction of plant express vector

Primer name	Primer sequence (5′-3′)	Tm (°C)	Product length (bp)
rd29A-F1	GGCTTTACACTTTATGCTTCC	49.2	859
rd29A-F2	TTGTTAGGCTCCCTCATTTC	49.2	859
rd29A-T1	CAGTTTGAAAGAAAAGGGAA	46.7	71
rd29A-T2	GCTTTTTGGAACTCATGTCG	46.7	71
MtDREB2A-F1	CATGCCATGGTGGAAATTGAAAGATGGGTGCT	53.5	971
MtDREB2A-F2	GGGTGACCGGATTATTATCTAGTTGCCCAAACG	53.5	971
MtDREB2A-T1	ACTTTTCCGACGGCTCAA	44.0	472
MtDREB2A-T2	GTCATTACACACACCCTCTC	44.0	472

表2 子叶节法转化大豆东农50的培养基成分

Table 2 List of components used in media preparation for transformation method

	Germination medium	Re-suspension medium	Co-cultivation medium	Shoot induction medium	Shoot elongation medium	Rooting medium
MS salts	1/2 ×	1/2 ×	-	-	-	-
MS iron stock	1/2 ×	1/2 ×	1 ×	1 ×	1 ×	1 ×
MS vitamins	1/2 ×	1/2 ×	-	-	-	-
B5 salts	-	-	1 ×	1 ×	1 ×	1/2 ×
B5 vitamins	-	-	1 ×	1 ×	1 ×	-
Sucrose (g·L^-1)	15	-	30	30	30	30
Glucose (g·L^-1)	-	10	-	-	-	-
Agar (g·L^-1)	8	-	5	8	8	8
pH	5.8	5.8	5.5	5.5	5.5	5.6
6-BA (mg·L^-1)	-	-	1.7	1.7	1.7	-
GA (mg·L^-1)	-	-	-	-	1.0	-
IBA (mg·L^-1)	-	-	-	-	-	1.0
MES (g·L^-1)	-	-	0.6	0.6	0.6	0.6
L-cys (mg·L^-1)	-	-	182.5	182.5	182.5	-
Na₂S₂O₃ (mg·L^-1)	-	-	250	250	250	-
DTT (mg·L^-1)	-	-	154.3	-	-	-
AS (mg·L^-1)	-	39.2	39.2	-	-	-
Cef (mg·L^-1)	-	-	-	100-400	100-400	-
Cb (mg·L^-1)	-	-	-	100-400	100-400	-
PPT (mg·L^-1)	-	-	-	0, 2-5	-	-

图1 植物表达载体pCAMBIA3301-rd29A-MtDREB2A的构建(A) rd29A启动子的PCR鉴定(1: Marker; 2: pCAMBIA3301-35S-GUS对照; 3-6: rd29A的PCR产物); (B) MtDREB2A基因的PCR鉴定(1: Marker; 2: pCAMBIA3301-35S-GUS对照; 3-6: MtDREB2A的PCR产物); (C) pCAMBIA3301-rd29A-MtDREB2A的结构示意图

Figure 1 Construction of plant express vector pCAMBIA3301-rd29A-MtDREB2A(A) PCR confirmation of rd29A promoter (1: Marker; 2: pCAMBIA3301-35S-GUS; 3-6: PCR product of rd29A); (B) PCR confirmation of MtDREB2A genes (1: Marker; 2: pCAMBIA3301-35S-GUS; 3-6: PCR product of MtDREB2A); (C) Structure of pCAMBIA3301-rd29A-MtDREB2A

表3 东农50种子NaClO法表面消毒实验结果(平均值±标准差)

Table 3 Effect of NaClO method on seed sterilization of Glycine max cv. ‘Dongnong 50’ (means±SD)

Treatment	NaClO concentration (%)	Time (min)	Sterilization rate (%)	Contamination rate (%)	Death rate (%)
1	0.10	3	81.33±3.06 e	16.00±2.00 b	2.67±1.16 bcd
2	0.10	5	72.67±1.16 f	24.00±2.00 a	3.33±1.16 bc
3	0.10	10	88.67±1.16 bc	10.67±1.16 cd	0.67±1.16 d
4	0.25	3	86.67±1.16 cd	10.00±2.00 cde	3.33±1.16 bc
5	0.25	5	70.67±2.31 f	23.33±4.16 a	6.00±2.00 a
6	0.25	10	90.67±1.16 b	8.00±2.00 de	1.33±1.16 cd
7	0.50	3	83.33±3.06 de	13.33±3.06 bc	3.33±1.16 bc
8	0.50	5	89.33±2.31 bc	6.00±2.00 ef	4.67±1.16 ab
9	0.50	10	98.67±1.16 a	0.67±1.16 g	0.67±1.16 d

表4 东农50种子Cl2气熏蒸法表面消毒实验结果

Table 4 Effect of Cl2 method on seed sterilization of Glycine max cv. ‘Dongnong 50’

Treatment	Time (h)	Sterilization rate (%)	Contamination rate (%)	Death rate (%)
1	1	82.00±5.29 b	13.33±5.78 a	4.66±1.16 b
2	2	82.67±6.11 b	12.00±6.00 a	5.33±3.06 b
3	4	92.00±3.46 a	2.67±2.31 b	5.33±3.06 b
4	6	93.33±1.16 a	2.67±2.31 b	4.00±2.00 b
5	8	88.00±3.46 ab	1.33±2.31 b	10.67±1.16 a

图2 GUS基因组织化学染色(A) 转化方法对GUS基因组织化学染色率的影响, 不同小写字母表示在0.05水平差异显著(Duncan’s test); (B), (C) 共培养后的子叶节GUS基因组织化学染色((B) 阴性对照; (C) 组织化学染色阳性)

Figure 2 GUS gene histochemical staining(A) The effect of transgenic methods on histochemical GUS staining rate, different lowercase letters indicate significant differences at 0.05 level according to Duncan’s test; (B), (C) The GUS gene histochemical staining of cotyledonary node after co-cultivation ((B) Negative control; (C) Positive result of histochemical stain)

表5 农杆菌侵染大豆东农50的条件优化

Table 5 The results of orthogonal experiment for Agrobacterium-mediated transformation of Glycine max cv. ‘Dongnong 50’

Treatment	Factor				Histochemical staining rate of cotyledonary-node (%)
Treatment	Germination time (d) (A)	Culture temperature (°C) (B)	OD₆₀₀ value (C)	Co-cultivation time (d) (D)	Histochemical staining rate of cotyledonary-node (%)
1	3	25	0.3	2	8.00±2.00 g
2	3	26	0.5	3	31.05±10.46 def
3	3	27	0.7	4	38.27±5.46 cde
4	3	28	0.9	5	46.30±17.65 bcd
5	4	25	0.5	4	39.70±19.34 cde
6	4	26	0.3	5	59.33±3.06 ab
7	4	27	0.9	2	4.00±2.00 g
8	4	28	0.7	3	21.33±6.43 efg
9	5	25	0.7	5	68.33±12.42 a
10	5	26	0.9	4	51.06±7.65 abc
11	5	27	0.3	3	30.56±16.17 def
12	5	28	0.5	2	4.00±5.29 g
13	6	25	0.9	3	40.95±11.61 bcd
14	6	26	0.7	2	8.52±1.70 g
15	6	27	0.5	5	20.36±9.30 efg
16	6	28	0.3	4	12.67±10.26 fg
K₁	30.91	39.25	27.64	6.13
K₂	31.09	37.49	23.78	30.97
K₃	38.49	23.30	34.12	35.43
K₄	20.62	21.07	35.58	48.58
R	17.87	18.18	11.80	42.45

图3 优化的子叶节法转化大豆东农50(A) 农杆菌侵染后共培养; (B) 不定芽诱导; (C) 不定芽伸长; (D) 抗性芽移栽成活; (E) 抗性植株GUS基因PCR检测(1: Marker; 2: pCAMBIA3301-35S-GUS; 3: 对照大豆; 4-13: 抗性植株); (F) 抗性植株GUS基因RT-PCR检测(1: Marker; 2: 对照大豆; 3-9: 抗性植株); (G) 对照大豆(左)和抗性植株(中、右)叶片的组织化学染色

Figure 3 Transformation system of Glycine max cv. ‘Dongnong 50’ using optimized cotyledonary-node method(A) Co-cultivation after transformation; (B) Shoot induction; (C) Shoot elongation; (D) Resistant bud transplant; (E) PCR confirmation of GUS gene (1: Marker; 2: pCAMBIA3301-35S-GUS; 3: Control soybean; 4-13: Resistant plant); (F) RT-PCR confirmation of GUS gene (1: Marker; 2: Control soybean; 3-9: Resistant plant); (G) Histochemical staining of leaves from control (left) and resistant plant (middle and right)

图4 转MtDREB2A基因大豆东农50鉴定和根系生长状况测定(A) MtDREB2A基因PCR检测(1: Marker; 2: pCAMBIA3301-rd29A-MtDREB2A; 3: 对照大豆; 4-6: 转MtDREB2A基因大豆); (B) 对照大豆根系(Bar=1 cm); (C) 对照和转MtDREB2A基因大豆主根长度; (D) 转MtDREB2A基因大豆荚果; (E) 转MtDREB2A基因大豆根系(Bar=1 cm); (F) 对照和转MtDREB2A基因大豆侧根数量。图(C)和(F)中不同小写字母表示在0.05水平差异显著(Duncan’s test)。

Figure 4 Confirmation and root system analyses of MtDREB2A transgenic soybean Dongnong 50(A) PCR result of MtDREB2A transgenic soybean (1: Marker; 2: pCAMBIA3301-rd29A-MtDREB2A; 3: Control soybean; 4-6: MtDREB2A transgenic soybean); (B) The root of control soybean (Bar=1 cm); (C) The length of taproot of control and transgenic soybeans; (D) The pod of MtDREB2A transgenic soybean in greenhouse; (E) The root of MtDREB2A transgenic soybean (Bar=1 cm); (F) The number of lateral root of control and transgenic soybeans. Different lowercase letters in Figures (C) and (F) indicate significant differences at 0.05 level according to Duncan’s test.

表6 不定芽诱导条件正交试验的不定芽诱导率和组织化学染色率

Table 6 The adventitious bud induction rate and histochemical staining rate of orthogonal experiment

Treatment	Factor					Adventitious bud induction rate (%)	Histochemical staining rate of adventitious bud (%)
Treatment	Recovery time (d) (A)		PPT concentration (mg·L^-1) (B)	Cef concentration (mg·L^-1) (C)	Cb concentration (mg·L^-1) (D)	Adventitious bud induction rate (%)	Histochemical staining rate of adventitious bud (%)
1	0		2	100	100	1.11±1.92 f	0.00±0.00
2	0		3	200	200	1.11±1.92 f	1.11±1.92
3	0		4	300	300	3.33±5.77 f	2.22±1.92
4	0		5	400	400	1.11±1.92 f	1.11±1.92
5	5		2	200	300	22.22±9.62 cd	2.22±1.92
6	5		3	100	400	7.78±3.85 ef	3.33±0.00
7	5		4	400	100	4.45±3.85 f	1.11±1.92
8	5		5	300	200	2.22±3.85 f	1.11±1.92
9	10		2	300	400	31.11±5.09 bcd	0.00±0.00
10	10		3	400	300	18.89±3.85 de	0.00±0.00
11	10		4	100	200	27.78±10.18 cd	0.00±0.00
12	10		5	200	100	21.11±8.39 cd	1.11±1.92
13	15		2	400	200	55.56±5.09 a	0.00±0.00
14	15		3	300	100	42.22±12.62 b	0.00±0.00
15	15		4	200	400	33.33±13.33 bc	0.00±0.00
16	15		5	100	300	23.33±8.82 cd	0.00±0.00
Adventitious bud induction rate	K₁	1.67	27.50	15.00	17.22
	K₂	9.17	17.50	19.44	21.67
	K₃	24.72	17.22	19.72	16.94
	K₄	38.61	11.94	20.00	18.33
	R	36.94	15.56	5.00	4.72
Histochemical staining rate	K₁	1.11	0.56	0.83	0.56
	K₂	1.94	1.11	1.11	0.56
	K₃	0.28	0.83	0.83	1.11
	K₄	0.00	0.83	0.56	1.11
	R	1.94	0.56	0.56	0.56

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