转Cry30Fa1基因抗褐飞虱水稻的获得及鉴定

doi:10.16819/j.1001-7216.2016.5190

摘要/Abstract

摘要：

将编码高效杀褐飞虱蛋白的苏云金芽胞杆菌基因Cry30Fa1密码子改造后,通过农杆菌介导法转入蜀恢818(R818),并最终获得46个转基因植株。通过定量PCR及Western Blot鉴定了Cry30Fa1在转录水平和蛋白水平的表达,并通过分子检测固定稳定表达的抗性基因,并结合传统育种系谱法选择具有优良农艺性状株系。对选育的株系在室内和大田环境下进行了抗虫性鉴定,选育的R818-Cry30Fal株系抗性明显优于亲本材料并达到抗的水平;在单株抗虫试验中,观察到了转基因株系对褐飞虱具有致死作用。说明转入Cry30Fa1基因使水稻产生了对褐飞虱抗性。培育出了具有抗褐飞虱蛋白的新型恢复系R818-Cry30Fal,为三系杂交育种提供了新的抗性材料并丰富了抗褐飞虱水稻种质资源。

关键词: 褐飞虱, 农杆菌介导法, 抗虫性, Cry30Fa1

Abstract:

Modified Cry30Fa1, a Bacillus thuringiensis (Bt) gene encoding a high-efficiency insecticidal protein to the brown planthopper(BPH),was introduced into Shuhui 818(R818) by Agrobacterium-mediated genetic transformation and 46 transgene lines were obtained. We confirmed the valid transformation by RT-qRCR and Western blot at transcriptional level and translational level, and fixed the stable expression Cry30Fa1 in homozygosis. The classical pedigree breeding methods were also used to select the important agronomic traits in transgenic lines. Furthermore, we evaluated the resistance to BPH of different lines in growth chamber and field, and found that the R818-Cry30Fal displayed higher resistance than untransformed R818 in seedling stage. We also observed the reduced population of BPH feeding the transgenic lines, suggesting that the transformed Cry30Fa1 enhanced resistibility to BPH in rice. We cultivated new BT protein transgenic rice restorer line R818-Cry30Fal, which may provide new BPH resistance material to three line hybrid rice breeding and enrich BPH resistance germplasm resources in rice.

Key words: brown planthopper, Agrobacterium-mediated method, insect resistance, Cry30Fa1

中图分类号:

王海鹏, 黄晓西, 梁越洋, 朱军, 张翠霞, 王秀梅, 贡常委, 郑爱萍, 邓其明, 李双成, 王玲霞, 李平, 王世全. 转Cry30Fa1基因抗褐飞虱水稻的获得及鉴定[J]. 中国水稻科学, 2016, 30(3): 256-264.

Hai-peng WANG, Xiao-xi HUANG, Yue-yang LIANG, Jun ZHU, Cui-xia ZHANG, Xiu-mei WANG, Chang-wei GONG, Ai-ping ZHENG, Qi-ming DENG, Shuang-cheng LI, Ling-xia WANG, Ping LI, Shi-quan WANG. Development and Identification of Insect Resistant Transgenic Rice with Cry30Fa1 Gene[J]. Chinese Journal OF Rice Science, 2016, 30(3): 256-264.

图/表 10

图1 载体pCDMAR-Cry30Fa1-Hyg的骨架

Fig.1. Skeleton of vector pCDMAR-Cry30Fa1-Hyg.

图2 转基因转化的整个过程 A-成熟胚的诱导; B-愈伤组织的继代; C-抗性愈伤的筛选; D-抗性愈伤的分化; E-抗性植株的生根。

Fig. 2. Procedure of transformation of rice. A, Induction of seed; B, Subculture of callus; C, Screening of resistant callus; D, Regeneration of resistance callus; E, Rooting of resistance callus.

图3 再生植株潮霉素T0代抗性检测 1-阴性对照; 2,3-未转进潮霉素基因的植株; 4-阳性对照; 5~7-转入潮霉素基因的植株。

Fig. 3. Response of leaves from transgenic plants T0 to hygromycin solution. 1, Leaves of non-transgenic rice; 2,3, Leaves of non-Hyg transgenic rice; 4, Positive control; 5,6,7, Leaves of Hyg transgenic rice.

图4 T0转化植株Hyg基因的PCR检测 M-DNA 标记,“+”表示以质粒pCDMAR-Cry30Fa1作为阳性对照,“-”表示以未进行转化的蜀恢818作为阴性对照;1-40为转入Hyg的T0代植株。

Fig. 4. Confirmation of Hyg gene in T0 generation by PCRz M, DNA marker; “+”, Plasmid of pCDMAR-Cry30Fa1; “-”, Non-transgenic Shuhui 818; 1-40, T0 transformants carring Hyg.

图5 T0转化植株Cry30Fa1基因的PCR检测 M-DNA 标记;“+”-质粒pCDMAR-Cry30Fa1作为阳性对照,“-”-未进行转化的蜀恢818作为阴性对照;1~40-转入Cry30Fa1的T0代植株。

Fig. 5. Confirmation of Cry30Fa1 gene in T0 plants by PCR. M, DNA marker; +, Plasmid of pCDMAR-Cry30Fa1; -, Non-transgenic Shuhui 818; 1-40, T0 transformants carring Cry30Fa1.

表1 转基因材料的主要农艺性状(平均值±标准差,n=3)

Table 1 Major agronomic traits of transgenic materials(mean±SD, n=3).

性状 Traits	蜀恢818 Shuhui 818	株系103 Line 103	株系129 Line 129	株系131 Line 131
株高 Plant height/cm	115.0±0.6 a	99.3±2.0 c	113.3±2.4 a	105.7±0.9 b
有效分蘖数 Effective tiller number	7.0±1.5 a	8.0±2.0 a	7.3±0.9 a	6.7±0.9 a
主穗长 Main panicle length/cm	21.4±0.4 a	22.6±0.5 a	24.6±0.3 b	23.8±0.3 b
结实率 Seed-setting rate/%	70.7±3.0 a	78.1±1.3 a	79.8±3.9 a	77.5±2.1 a
千粒重 1000-grain weight/g	25.4±1.2 a	27.0±0.6 a	27.6±0.2 a	25.0±1.3 a

图6 Cry30Fa1在灌浆期叶片中的表达情况 **在0.01水平差异显著。

Fig. 6. Expression level of Cry30Fa1 in leaf at the filling stage. **,Significant difference at 0.01 level.

图7 Cry30Fa1在灌浆期叶片中的表达情况 ns-无显著差异; **在0.01水平差异显著。

Fig. 7. Expression of Cry30Fa1 in leaf at the filling stage. ns,No significant difference;**,Significant difference at 0.01 level.

图8 三种不同抗虫鉴定方法的鉴定结果 A-单株抗虫性鉴定; B-苗期集团抗虫鉴定; C-大田抗虫性鉴定。

Fig. 8. Pest resistance identification results by three different methods. A, Individual identification of pest resistance; B, Pest resistance identification in seedling group; C, Field identification of pest resistance.

表2 不同鉴定方法下水稻抗虫性的分析(平均值±标准差,n=3)

Table 2 Insect resistance of rice by different identification methods(mean± SD, n=3).%

鉴定方式 Identification method	蜀恢818 Shuhui 818	株系985 Line 985	株系103 Line 103	株系129 Line 129	株系131 Line 131
集团鉴定 Group identification
水稻死亡率 Rice mortality	81.1±3.1 a	52.7±7.4 bc	51.0±1.7 c	61.7±2.5 b	52.8±3.0 bc
单株鉴定 Plant identification
褐飞虱死亡率 BPH mortality	21.3±8.6 b	28.0±8.2 ab	31.0±9.5 a	30.3±8.3 a	34.0±11.5 a
田间鉴定 Field identification
水稻死亡率 Rice mortality	64.2±6.0 a	0±0 c	29.2±4.4	3.5±6.6 b	16.7±4.4 b

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