发根农杆菌介导的菜用豌豆遗传转化体系构建

doi:10.16420/j.issn.0513-353x.2023-0944

摘要/Abstract

摘要：

创建了一套由发根农杆菌介导，简单高效的菜用豌豆遗传转化体系。以播种于无菌基质中菜用豌豆‘浙豌1号’为材料，以在子叶节和下胚轴连接处切割后的上部幼苗为外植体，用导入pCAMBIA3301-GUS载体的发根农杆菌AR1193诱导外植体生成毛状根，毛状根诱导率为100%。7 d苗龄幼苗更有利于毛状根的诱导，平均生根数为14.60。对毛状根进行GUS基因RT-PCR检测及GUS染色验证，发现外源GUS基因可在毛状根中成功表达，毛状根转化率为35.21%。

关键词: 菜用豌豆, 发根农杆菌, 苗龄, 毛状根, 遗传转化, 转基因根

Abstract:

This study established a simple and efficient Agrobacterium rhizogenes-mediated genetic transformation system in vegetable pea. The vegetable pea seeds of‘Zhewan1’were sown into pots with wet sterile vermiculite. The hypocotyls of vegetable pea seedlings were cut at the junction of cotyledon node and hypocotyl and the residual seedlings were selected as explants. The hairy roots were induced with Agrobacterium rhizogenes AR1193 strain harboring the construct of pCAMBIA3301-GUS. The results indicated that 100% of the inoculated explants could produce hair roots. The effect of different seedling age of explant on hairy root induction was also investigated. It was found that the 7-day-old seedling was favorable for hair root induction and the number of hairy root per seedling was 14.60. Further，the hair roots were amplified by RT-PCR of GUS gene and stained with the GUS dye. It was proven that the exogenous GUS gene could be successfully expressed in the hair roots and 35.21% of the hair roots were transgenic positive roots.

Key words: vegetable pea, Agrobacterium rhizogenes, seeding age, hairy root, genetic transformation, transgenic root

冯志娟, 刘娜, 卜远鹏, 张古文, 王斌, 龚亚明. 发根农杆菌介导的菜用豌豆遗传转化体系构建[J]. 园艺学报, 2024, 51(10): 2439-2448.

FENG Zhijuan, LIU Na, BU Yuanpeng, ZHANG Guwen, WANG Bin, GONG Yaming. Establishment of Agrobacterium rhizogenes-Mediated Genetic Transformation System in Vegetable Pea[J]. Acta Horticulturae Sinica, 2024, 51(10): 2439-2448.

图/表 6

图1 菜用豌豆幼苗在基质中的生长状态

Fig. 1 Seedlings of vegetable pea cultivated in the vermiculite

图2 发根农杆菌介导的菜用豌豆遗传转化过程 A：适宜转化的苗龄（7 d）；B：携带载体pCAMBIA3301-GUS的发根农杆菌AR1193固体TY平板培养基；C：携带载体pCAMBIA3301-GUS的发根农杆菌AR1193的TY菌液；D：切掉子叶和主根后上部幼苗外植体产生伤口裹菌；E：已裹菌的外植体种植于无菌基质后浇TY菌液；F：塑料罩保湿培养；G：诱导形成的毛状根；H：毛状根GUS染色。

Fig. 2 Agrobacterium rhizogenes-mediated genetic transformation process of vegetable pea A：Ideal stage（7-day-old seedling）of vegetable pea for transformation；B：Agrobacterium rhizogenes AR1193 strain containing the pCAMBIA3301-GUS vector cultured on the solid TY plate；C：Agrobacterium rhizogenes AR1193 strain containing pCAMBIA3301-GUS vector cultured in the liquid TY medium；D：The cotyledon and primary root were cut and the cut of residual seedling was scraped on the solid plate grown AR1193；E：Inoculated explants（coated the bacterial mass）were planted into sterile vermiculite and watered with liquid TY medium；F：Inoculated explants were covered with the plastic cover；G：Induced hairy roots；H：GUS staining of hairy roots.

图3 不同苗龄（5、7和9 d）菜用豌豆外植体侵染AR1193-pCAMBIA3301菌株后不同天数（10、14和18 d）对诱导毛状根的影响

Fig. 3 Effect of different seedling age（5，7 or 9 d）of explant inoculated with AR1193-pCAMBIA3301 strain for different treatment time（10，14 or 18 d）on hairy root induction

表1 不同苗龄菜用豌豆外植体毛状根诱导效率

Table 1 Induction efficiency of hairy root from different seedling age of explant

苗龄 Seedling stage	培养时间 Cultivation time	侵染外植体数 Number of the explant inoculated	产生毛状根外植体数 Number of explant produced hairy root	外植体平均生根数 Number of hairy root per explant	GUS染色显蓝毛状根数 Number of GUS positive root per explant	毛状根诱导率/% Induction efficiency of hairy root	毛状根转化率/% Transformation frequency of hairy root
5 d	10 d	30	30	3.00 ± 1.61 d		100
	14 d	30	30	4.00 ± 1.70 cd		100
	18 d	30	30	6.50 ± 1.80 c		100
7 d	10 d	30	30	6.50 ± 2.20 c	5.14 ± 1.55	100	35.21
	14 d	30	30	14.60 ± 1.72 a		100
	18 d	30	30	14.60 ± 2.40 a		100
9 d	10 d	30	30	6.50 ± 1.62 c		100
	14 d	30	30	11.00 ± 2.60 b		100
	18 d	30	30	11.00 ± 2.10 b		100

图4 转基因菜用豌豆毛状根GUS染色验证

Fig. 4 Validation of transgenic hairy roots of vegetable pea by GUS staining

图5 转基因菜用豌豆毛状根RT-PCR验证 M：DNA marker；1 ~ 5：AR1193-pCAMBIA3301-GUS侵染后转基因毛状根；6：pCAMBIA3301-GUS质粒；7 ~ 9：未侵染根；10：AR1193菌株；11：H2O。

Fig. 5 Validation of transgenic hairy roots of vegetable pea by RT-PCR M：DNA marker；1-5：Transgenic hairy roots inoculated by AR1193-pCAMBIA3301-GUS；6：pCAMBIA3301-GUS plasmid；7-9：Non-inoculated roots；10：AR1193 strain；11：H2O.

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