转昆虫抗冻蛋白基因增强甘薯抗冻能力

doi:10.11983/CBB19133

摘要/Abstract

摘要： 为明确昆虫抗冻蛋白基因转入甘薯(Ipomoea batatas)后是否能提升其抗冻能力, 进而为培育甘薯抗冻育种材料奠定基础, 将黄粉虫(Tenebrio molitor)抗冻蛋白基因TmAFP导入植物基因表达质粒, 经农杆菌介导的遗传转化获得抗冻甘薯新材料。以甘薯品种Huachano为受体材料建立甘薯植株高效再生体系, 并采用不同成分的体细胞胚成熟培养基培养胚性悬浮细胞。胚性愈伤组织对除草剂的敏感性测试结果表明, 转基因阳性植株筛选的最适培养基为MS+0.2 mg·L ^-12,4-D+0.8 mg·L ^-1GAP+100 mg·L ^-1Carb。将表达质粒分别转化Huachano后共获得7个胚性愈伤团并最终获得42株再生抗性植株, 其中转pSUIBEV3-AFP有23个株系, 转pCAMBIA-AFP有19个株系, 经PCR、Southern杂交和RT-PCR检测后证实TmAFP基因已整合至甘薯基因组中并获得表达。将转基因甘薯及对照植株在-1°C下处理15小时后转移至室温, 结果表明, 转基因甘薯植株的抗冻能力显著提升。

关键词: 甘薯, 抗冻蛋白, 转基因, 甘薯植株再生, 分子育种

Abstract: To explore whether the gene encoding antifreeze protein from insect can enhance the freezing tolerance of sweet potato through gene transformation, and to prepare freeze-tolerance materials for breeding purposes, we constructed a plant gene expression vector harboring an antifreeze gene TmAFP from yellow mealworms (Tenebrio molitor) and obtained transgenic freeze-tolerance sweet potato lines using Agrobacterium-mediated transformation method. A high-frequency regeneration system of sweet potato was established using the variety Huachano as the recipient material, and the embryogenic suspension cells were cultured in the somatic embryo maturation medium. The sensitivity test of embryogenic cells to herbicides indicated that the combination of MS+0.2 mg·L ^-12,4-D+0.8 mg·L ^-1GAP+100 mg·L ^-1Carb is the most effective medium for screening the transgenic positive plants. Seven embryogenic calli were obtained and 42 resistant seedlings were regenerated, among which 23 harbored pSUIBEV3-AFP and 19 had pCAMBIA-AFP. All resistant seedlings were examined by PCR, Southern hybridization and RT-PCR, and the results showed that the TmAFP gene was integrated into the plant genome and expressed. The transgenic and non-transgenic plants were treated at -1°C for 15 hours, and then transferred to room temperature. The results demonstrated that the freeze-tolerance of the transgenic plants was greatly improved.

Key words: sweet potato, antifreeze protein, transgenic, plant regeneration of sweet potato, molecular breeding

赖先军,张义正,古英洪,颜朗. 转昆虫抗冻蛋白基因增强甘薯抗冻能力. 植物学报, 2020, 55(1): 9-20.

Xianjun Lai,Yizheng Zhang,Yinghong Gu,Lang Yan. Transformation of Insect Derived Antifreeze Gene into Sweet Potato (Ipomoea batatas) and Enhanced Its Freeze-tolerance. Chinese Bulletin of Botany, 2020, 55(1): 9-20.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB19133

https://www.chinbullbotany.com/CN/Y2020/V55/I1/9

图/表 6

图1 甘薯遗传转化体系 (A), (B) 分别在MS+0.2 mg·L-1 2,4-D液体培养基中继代18和22周后的甘薯胚性悬浮细胞; (C) 在MS+0.2 mg·L-1 2,4-D固体培养基上培养8周后的甘薯胚性悬浮细胞; (D) 甘薯体细胞胚再生的小植株。(A), (B), (D) Bars=1 cm; (C) Bar=4 cm

Figure 1 Transformation system of sweet potato (A), (B) Sweet potato embryogenic suspension cells at 18 and 22 weeks cultivated in MS+0.2 mg·L-1 2,4-D liquid medium, respectively; (C) Sweet potato embryogenic suspension cells at 8 weeks cultivated in MS+0.2 mg·L-1 2,4-D solid medium; (D) Sweet potato seedlings regenerated from somatic embryo. (A), (B), (D) Bars=1 cm; (C) Bar=4 cm

表1 不同体细胞胚成熟培养基对甘薯芽苗再生的影响

Table 1 Effect of different somatic embryo maturation medium on the regenerated seedlings

Treatment	Components (mg·L^-1)	Number of seedlings in average	*	**
1	MS	44.8±5.22	a	A
2	MS+ABA1.0	33.6±5.18	b	AB
3	MS+GA₃1.0	30.2±4.73	bc	B
4	MS+ABA1.0+GA₃1.0	23.2±6.29	c	B
5	MS+ABA4.0+GA₃1.0	21.8±6.58	c	B

图2 不同浓度除草剂和抗生素对甘薯胚性愈伤组织和组培苗的影响 (A) 选择培养基上培养8周的甘薯胚性悬浮细胞。从左到右培养基依次为MS+0.2 mg·L-1 2,4-D分别添加0、0.2、0.4、0.6和0.8 mg·L-1 GAP; (B) 培养6周(从左至右第1, 2)和8周(3, 4)的胚性悬浮细胞, 培养基分别为MS+0.2 mg·L-1 2,4-D (1, 3)和MS+0.2 mg·L-1 2,4-D+100 mg·L-1 Carb (2, 4); (C) 培养8周的胚性悬浮细胞, 培养基为MS+0.2 mg·L-1 2,4-D+100 mg·L-1 Carb分别添加0.8、1.0、1.2和1.4 mg·L-1 GAP; (D) 分别为MS+0.2 mg·L-1 2,4-D+0.8 mg·L-1 GAP+100 mg·L-1 Carb培养基上接种0天和培养3周的Huachano茎尖。(A), (B), (C) Bars=1 cm; (D) Bar=2 cm

Figure 2 Effects of herbicide and antibiotic in different concentrations on embryogenic callus and regenerated seedlings of sweet potato (A) Sweet potato embryogenic suspension cells at 8 weeks cultivated in selective medium. The medium from left to right was MS+0.2 mg·L-1 2,4-D with 0, 0.2, 0.4, 0.6, 0.8 mg·L-1 GAP, respectively; (B) Embryogenic suspension cells at 6 weeks (the first and second from left to right) and 8 weeks (the third and fourth). The medium were MS+0.2 mg·L-1 2,4-D (the first and third) MS+0.2 mg·L-1 2,4-D+100 mg·L-1 Carb (the second and fourth); (C) Embryogenic suspension cells at 8 weeks, the medium are MS+0.2 mg·L-1 2,4-D+100 mg·L-1 Carb with 0.8, 1.0, 1.2, 1.4 mg·L-1 GAP, respectively; (D) Huachano stem tips cultivated 0 day and 3 weeks on medium of MS+0.2 mg·L-1 2,4-D+0.8 mg·L-1 GAP+100 mg·L-1 Carb, respectively. (A), (B), (C) Bars=1 cm; (D) Bar=2 cm

图3 甘薯抗性体胚及其转基因植株再生过程 (A) 用于转化的甘薯胚性悬浮细胞在MS+0.2 mg·L-1 2,4-D液体培养基中28°C黑暗振荡培养32周; (B) 选择与非选择培养4周的胚性愈伤组织, a-c为选择性培养基(MS+0.2 mg·L-1 2,4-D+100 mg·L-1 Carb+0.8 mg·L-1 GAP), 箭头所指为抗性愈伤组织; d为未加除草剂的对照; (C) 转基因植株再生过程, a: 再生组织; b: 生芽; c: 生叶; d: 成株; e: 成苗。Bars=1 cm

Figure 3 Sweet potato resistant somatic embryo and the regeneration of transgenic plants (A) Sweet potato embryogenic suspension cells cultivated in MS+0.2 mg·L-1 2,4-D liquid medium at 28°C for 32 weeks; (B) Embryogenic callus cultivated in selective and non-selective medium for 4 weeks, a-c: Embryogenic callus cultivated in selective medium (MS+0.2 mg·L-1 2,4-D+100 mg·L-1 Carb+0.8 mg·L-1 GAP), resistant callus was marked by arrows; d: Embryogenic callus cultivated in control medium without herbicide; (C) The processes of transgenic plant regeneration, a: Reproductive tissue; b: Bud; c: Leaf; d: Seedling; e: Reproductive plant. Bars=1 cm

图4 TmAFP转基因甘薯植株的鉴定 (A) 采用除草剂(1.0 mg·L-1 GAP)对再生植株进行筛选(4-12、4-11、4-10、4-9: 抗性植株; 47-1、47-2: 非抗性植株; CK: 非转基因对照); (B) PCR快速鉴定转基因植株(AFP片段长度353 bp) (M: D2000 DNA marker (下同); +CK: pSUIBEV3-AFP质粒正对照; -CK: 未转化对照植株; 4-9、4-10、4-12: GAP抗性植株; 47-2: 非抗性植株); (C) 转基因植株的PCR检测(+CK: pSUIBEV3-AFP质粒作正对照扩增AFP基因; -CK: 未转化对照植株扩增AFP基因; 4-9-AFP: 转基因植株扩增AFP基因; 4-9-RPB2: 转基因植株扩增RPB2基因; -CK-RPB2: 未转化对照植株扩增RPB2基因); (D) Southern杂交(+CK: pCAMBIA-AFP质粒正对照; -CK: 未转化对照植株; 其余为不同转基因株系); (E) 转基因植株RT-PCR检测(RP1、RP2: 转pCAMBIA空载植株; 4-12、4-11、44-1、54-5: 转pCAMBIA-AFP株系)。

Figure 4 Detection of TmAFP in the transgenic sweet potato plants (A) Screening with 1.0 mg·L-1 GAP (Line 4-12, 4-11, 4-10, 4-9: Resistant seedlings; Line 47-1, 47-2: Non-resistant seedlings; CK: Non-transgenic control); (B) Amplified 353-bp fragment of AFP gene (M: D2000 molecular weight marker; +CK: pSUIBEV3-AFP vector as positive control; -CK: Non-transgenic seedlings as negative control; Line 4-9, 4-10, 4-12: GAP resistant seedlings; Line 47-2: Non-resistant seedlings); (C) PCR detection of transgenic seedlings (+CK: Amplifying AFP gene using pSUIBEV3-AFP as template; -CK: Amplifying AFP gene using non-transgenic seedling; 4-9-AFP: Amplifying AFP gene in transgenic seedling; 4-9-RPB2: Amplifying RPB2 gene in transgenic seedling; -CK-RPB2: Amplifying RPB2 gene in non-transgenic seedling); (D) Southern blotting analysis (+CK: pCAMBIA-AFP vector as positive control; -CK: Non-transgenic seedlings as negative control; The others represent different transgenic seedling lines); (E) RT-PCR detection of transgenic seedlings (RP1, RP2: Transgenic seedlings with empty pCAMBIA vector; Line 4-12, 4-11, 44-1, 54-5: Transgenic seedlings with pCAMBIA-AFP vector).

图5 TmAFP转基因甘薯植株抗冻能力的检测 (A) 不同冷处理条件下植株电导率变化(CK1: 非转基因对照; CK2-4: 转空载的非抗冻植株; 4-9、4-10、4-11、4-12、44-1和54-5: TmAFP转基因株系; ** 表示差异极显著(P<0.01)); (B) -1°C条件下处理15小时后转移至室温条件, 转基因甘薯植株及对照的表型变化(Overall: 盆中同时栽种CK1、4-9、4-10、4-11、4-12; Non-transgenic: CK1放大图; Transgenic: 转基因株系4-9放大图)。Bars=5 cm

Figure 5 Detection of freezing-tolerance ability of TmAFP transgenic sweet potato plants (A) Conductivity assay under different freeze-treatments (CK1: Non-transgenic control; CK2-4: Transgenic plants with empty vector; 4-9, 4-10, 4-11, 4-12, 44-1, 54-5: TmAFP transgenic lines; ** indicate extremely significant differences (P<0.01)); (B) Phenotypic changes of transgenic sweet potato plants and controls after 15 h treatment at -1°C (Overall: CK1, 4-9, 4-10, 4-11, 4-12 planted in the same pot; Non-transgenic: Zoomed in CK1; Transgenic: Zoomed in transgenic line 4-9). Bars=5 cm

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