发根农杆菌介导的甘薯遗传转化体系优化

doi:10.13560/j.cnki.biotech.bull.1985.2023-0369

摘要/Abstract

摘要：

遗传转化体系对植物基因功能的验证具有重要意义，为在甘薯（Ipomoea batatas L.）中建立发根农杆菌（Agrobacterium rhizogenes）介导的遗传转化体系，本研究采用3×3×5三因素试验设计研究发根农杆菌侵染时间、外植体类型、发根农杆菌菌株对甘薯毛状根诱导率的影响。确定合适的外植体类型、侵染时间，在此基础上采用5×5双因子设计，研究5种发根农杆菌菌株和甘薯品种（系）对甘薯发根诱导率的影响。用PCR技术检测诱导出的毛状根，证实了发根农杆菌 Ri质粒的rolB 基因已经整合到甘薯细胞基因组中。结果表明，发根农杆菌侵染时间、外植体类型、发根农杆菌菌株、甘薯品种均影响发根诱导率，且各因子间存在交互作用。最适宜发根的外植体为茎段，最佳侵染时间是20 min，‘徐薯22’适宜菌株为MSU440，‘泰中6号’适宜菌株为K599，‘1610’适宜菌株为C58C1，‘YS’适宜菌株为K599，‘灰薯’适宜菌株为C58C1。采用发根农杆菌菌株MSU440，以‘徐薯22’茎段为外植体、农杆菌侵染20 min，最高获得了79.63%的毛状根诱导率。构建过表达载体进行毛状根遗传转化，通过目的基因的PCR 扩增发现甘薯毛状根的转基因效率为38.1%。建立了发根农杆菌介导的甘薯遗传转化体系，为甘薯进一步的转基因育种奠定基础。

关键词: 甘薯, 发根农杆菌, 毛状根, 诱导率, 转基因根

Abstract:

Genetic transformation system is of great significance for the verification of plant gene function. In order to establish Agrobacterium rhizogenes mediated genetic transformation system in sweet potato（Ipomoea batatas L.）, a three-factor design of 3×3×5 was used to study the effects of infection time, explant type, and A. rhizogenes strains on the induction rate of hairy roots in sweet potato. The appropriate explant type and infection time were determined, and the 5×5 two-factor design was used to study the effects of five A. rhizomei strains and sweet potato varieties（lines）on the induction rates of sweet potato. The detection of induced hairy roots by PCR confirmed that the rolB gene of the A. rhizogenes Ri plasmid was integrated into the genome of sweet potato cells. The results showed that the infection time, explant type, A. rhizogenes strain, and sweet potato variety all affected the induction rate of hairy roots, and there was an interaction between the factors. Stem segment was the most suitable explants for hair roots, and the optimal infection time was 20 min. MSU440 was the suitable strain for ‘Xushu 22’. The suitable strain for ‘Taizhong 6’ ‘YS’ were K599. The suitable strain for‘1610’ was C58C1. The suitable strain for ‘Huishu’was C58C1. Using the A. rhizogenes strain MSU440, stem segments of ‘Xushu 22’were used as explants and Agrobacterium infection for 20 min, and the highest induction rate of hairy root was 79.63%. The transgenic efficiency of the hairy root of sweet potato was found to be 38.1% by PCR amplification of the target gene. A genetic transformation system of sweet potato mediated by A. rhizogenes was established, which lays the foundation for further transgenic breeding of sweet potato.

Key words: sweet potato, Agrobacterium rhizogenes, hairy root, induction rate, transgenic root

陶娜, 李茂兴, 郭华春. 发根农杆菌介导的甘薯遗传转化体系优化[J]. 生物技术通报, 2023, 39(10): 175-183.

TAO Na, LI Mao-xing, GUO Hua-chun. Optimization of Sweet Potato Genetic Transformation System Mediated by Agrobacterium rhizogenes[J]. Biotechnology Bulletin, 2023, 39(10): 175-183.

图/表 9

表1 发根农杆菌类型及抗性

Table 1 A. rhizogenes strains and their resistances

发根农杆菌菌株 Agrobacterium strain	冠瘿碱类型 Crown gallin type	抗性 Resistance
Ar.1193	农杆碱型 Agropine	链霉素 Streptomycin
K599	黄瓜碱型 Cucumopine	链霉素 Streptomycin
C58C1	农杆碱型 Agropine	链霉素 Streptomycin
ArQual	农杆碱型 Agropine	链霉素 Streptomycin
MSU440	农杆碱型 Agropine	链霉素 Streptomycin

表2 引物列表

Table 2 List of primers

引物名称 Primer name	引物序列 Primer sequences（5'-3'）	产物长度 Product length/bp
rolB-F	AAGTGCTGAAGGAACAATC	585
rolB-R	CAAGTGAATGAACAAGGAAC	585
HBFD1-F	ATGGCTCCTGTTCTCCTTGG	1 224
HBFD1-R	CGCTCCTTGTGATGCTCG	1 224

图1 不同菌株诱导不同外植体对毛状根的影响 A: Ar.1193; B: ArQual; C: C58C1; D: K599; E: MSU440

Fig. 1 Effects of different explants induced by different strains on hairy roots

图2 不同菌株和侵染时间对毛状根影响不同小写字母表示处理间差异显著（P<0.05）。下同

Fig. 2 Effects of different strains and infection times on hairy roots Different lowercase letters indicate a significant difference between treatments （P <0.05）. The same below

图3 不同菌株对不同甘薯品种（系）毛状根诱导率A-E分别为发根农杆菌对‘徐薯22’‘泰中6号’‘1610’（品系）‘灰薯’‘YS’（品系）的毛状根诱导情况。下同

Fig. 3 Hairy roots induction rates of different sweet potato varieties（lines） by different strains A-E shows the hairy root induction of ‘Xushu 22’ ‘Taizhong No. 6’ ‘1610’ ‘Huishu’ and ‘YS’. The same below

图4 不同甘薯品种（系）发根诱导率

Fig. 4 Hairy root induction rates of different sweet potato varieties（lines）

表3 发根农杆菌类型和甘薯品种（系）对毛状根诱导率

Table 3 Type of Agrobacterium rhizogenes and sweet potato varieties（lines）

发根农杆菌菌株 Agrobacterium rhizogenes strain	甘薯品种（系）Sweet potato varieties（lines）
发根农杆菌菌株 Agrobacterium rhizogenes strain	泰中6号 Taizhong No. 6	1610 1610	徐薯22 Xushu22	YS YS	灰薯 Huishu	平均诱导率 Mean induction rate
Ar.1193	56.85±4.38B	25.93±2.26EF	43.34±0.68CD	10.39±0.46HI	12.81±2.0GH	29.86
K599	59.19±4.51B	48.73±3.09BC	75.24±5.24A	25.43±1.70EF	15.46±1.67FG	44.81
C58C1	58.89±4.84B	57.16±4.76B	11.80±1.28HI	19.31±1.60FG	20.0±0.00FG	33.43
ArQual	42.94±3.84CD	22.26±1.73FG	79.40±1.29A	19.0±2.58FG	14.02±2.04FG	35.52
MSU440	36.53±4.28DE	24.60±2.75FG	79.63±1.85A	5.69±0.29I	18.38±2.38FG	32.97
平均诱导率 Average induction rate	50.88	35.74	57.89	15.97	16.13

图5 甘薯毛状根rolB基因PCR检测图 1、2为甘薯的未转化根，作为阴性对照，3-7为Ar.1193、K599、C58C1、ArQual、MSU440侵染的甘薯茎段诱导出的毛状根，下同

Fig. 5 PCR detection of rolB gene in sweet potato hairyroots 1 and 2 are untransformed roots of sweet potato, as negative controls. 3-7 are the hairy roots induced by the stem segments of sweet potato infected by Ar. 1193, K599, C58C1, ArQual, and MSU440. The same below

图6 甘薯毛状根HBFD1基因PCR检测图

Fig. 6 PCR detection of HBFD1 gene in sweet potato hairyroots

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