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作物学报 ›› 2021, Vol. 47 ›› Issue (5): 789-798.doi: 10.3724/SP.J.1006.2021.04169

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

新型抗广谱性除草剂草甘膦转基因油菜的创制及其鉴定

李杰华1,3(), 端群1,3(), 史明涛1,3(), 吴潞梅1,3, 柳寒2, 林拥军1, 吴高兵3, 范楚川1,3,*(), 周永明1,3,*()   

  1. 1华中农业大学作物遗传改良国家重点实验室, 湖北武汉 430070
    2浙江省农业科学院, 浙江杭州 310021
    3华中农业大学植物科学技术学院, 湖北武汉 430070
  • 收稿日期:2020-07-25 接受日期:2020-11-01 出版日期:2021-05-12 网络出版日期:2020-12-14
  • 通讯作者: 范楚川,周永明
  • 作者简介:李杰华, E-mail: 1023629768@qq.com|端群, E-mail: 554246233@qq.com|史明涛, E-mail: 843121993@qq.com
  • 基金资助:
    国家转基因生物新品种培育重大专项(2018ZX08020001)

Development and identification of transgenic rapeseed with a novel gene for glyphosate resistance

LI Jie-Hua1,3(), DUAN Qun1,3(), SHI Ming-Tao1,3(), WU Lu-Mei1,3, LIU Han2, LIN Yong-Jun1, WU Gao-Bing3, FAN Chu-Chuan1,3,*(), ZHOU Yong-Ming1,3,*()   

  1. 1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, Hubei, China
    2Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, Zhejiang, China
    3College of Plant Science & Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2020-07-25 Accepted:2020-11-01 Published:2021-05-12 Published online:2020-12-14
  • Contact: FAN Chu-Chuan,ZHOU Yong-Ming
  • Supported by:
    National Major Project for Developing New GM Crops(2018ZX08020001)

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摘要：

草甘膦是世界上应用最广泛的广谱性除草剂, 目前我国还没有自主知识产权的抗草甘膦油菜品种。本研究利用农杆菌介导的油菜下胚轴遗传转化方法, 将新型抗草甘膦基因I. variabilis EPSPS转入甘蓝型油菜品系J9707中, 获得了126株阳性转化株, 阳性率为97.0%。这些转化单株中的T-DNA插入以单拷贝为主(占44.8%)。通过反向PCR确定了EPS-2、EPS-6和EPS-7等油菜转化体中T-DNA插入位置, 并设计转化体特异性引物对它们的T0~T3代材料进行检测, 证明了它们的T-DNA在基因组水平上整合的稳定性。RNA和蛋白水平的表达分析证实, I. variabilis EPSPS转基因及其蛋白产物在各转化株系不同世代能够稳定表达。苗期进行不同剂量的除草剂喷施处理发现, EPS-1、EPS-2、EPS-5、EPS-6和EPS-7等株系可耐受4倍田间推荐使用剂量的草甘膦。本研究所创建的新型抗草甘膦油菜种质资源将为我国抗除草剂油菜品种培育奠定了重要基础。

关键词: 甘蓝型油菜, I. variabilis EPSPS, 转基因, 草甘膦, 分子特征

Abstract:

Glyphosate is the most widely used broad-spectrum herbicide in the world. However, at present there is no glyphosate-tolerant rapeseed variety with independent intellectual property rights in China. In the study, a novel glyphosate-resistant genes I. variabilis EPSPS was transferred to the Brassica napus pure line J9707 via the Agrobacterium tumefaciens-mediated hypocotyl method, and 126 T0-positive transgenic plants with 97.0% positive rate were generated. The T-DNA insertion with a single copy (44.8%) is dominant. The insertion locations of T-DNA in the lines of EPS-2, EPS-6, and EPS-7 were identified by inverse PCR method. The stability of the T-DNA insertion in these lines were further confirmed by insertion-specific PCR in their T0 to T3 plants. The gene expression analysis revealed that the I. variabilis EPSPS gene and its protein was stably expressed in different generations of transgenic lines in RNA and protein levels. Treatments with different doses of glyphosate indicated that the lines of EPS-1, EPS-2, EPS-5, EPS-6, and EPS-7 could tolerate four times of the recommended dose of glyphosate in production. Thus, the novel glyphosate-tolerant rapeseed lines generated in the present study will lay the foundation for the herbicide- tolerance rapeseed breeding in China.

Key words: Brassica napus, I. variabilis EPSPS, transgene, glyphosate, molecular characteristics

图1

pTGH-1质粒的T-DNA示意图"

表1

本研究所用引物序列"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
EPSPS-F ATTAGCGCTAGGGACGTGAG P1 CTCTAGCCAATACGCAAACCGCC
EPSPS-R ATACGCTCCCACATCCTGTC P3 TGTTGTGTGGAATTGTGAGCGGA
35SP-1 GAAACCGGTTACAGGCAAATG SP2 CAGGTGGAGAACGTCGGTACGACT
35SP-2 AGAGGCGGTTTGCGTATT SP3 GGGTTTCGCTCATGTGTTGAGCA
35ST-1 ATAACACATTGCGGACGTTT BnACT-qRTF CTGGAATTGCTGACCGTATGAG
35ST-2 AGCCGGTGCTTGATAACT BnACT-qRTR ATCTGTTGGAAAGTGCTGAGGG
35SP-2 AGAGGCGGTTTGCGTATT EPs-qRTF CAGGGATGGACTCGTTATCAC
p130-35sF CAAGTGGATTGATGTGATAACATG EPs-qRTR GAGGGTCTTCGCAGTCGT
p130-35sR GTAGAGAGAGACTGGTGATTTCAGC EPSPS6 F GGCTTCTTAGTGGGCCTTTC
Iva-EPSPS-F ATTAGCGCTAGGGACGTGAG EPSPS7 F CATCCGCTTTACGACGAAACT
Iva-EPSPS-R ATACGCTCCCACATCCTGTC EPSPS2 F GAACTGAGCATGAATGGCATAAA
P5 ATGTGTGAGTAGTTCCCAGATAAGG

图2

转I. variabilis EPSPS基因植株的PCR阳性检测结果 A: 利用引物对EPSPS-F/EPSPS-R检测目的基因I. variabilis EPSPS的PCR结果; B: 利用引物对35SP-1/35SP-2检测CaMV 35S启动子元件的PCR结果; C: 利用引物对35ST-1/35ST-2检测CaMV 35S终止子元件的PCR结果。M为2 kb DNA marker; P为pTGH质粒(阳性对照); N为转化受体J9707 (阴性对照); 1~12为T0代部分转化植株, 编号分别为EPS-1~EPS-12。"

图3

转I. variabilis EPSPS基因T0代阳性植株的Southern blot拷贝数检测结果 A: Southern blot检测结果。杂交探针为位于目的基因上的EPSPS探针, 基因组DNA采用Hind III酶切。M: DNA marker; 1~31: 转基因阳性植株; 1: EPS-1; 3: EPS-2; 12: EPS-7; 29: EPS-3; 31: EPS-6; N: J9707 (阴性对照)。B: 不同拷贝数的单株统计。"

图4

T2和T3代转I. variabilis EPSPS基因植株的Southern blot杂交拷贝数检测 A: EcoR I酶切、CaMV35S promoter探针; B: Hind III酶切、CaMV35S promoter探针; C: EcoR I酶切、EPSPS探针; D: Hind III酶切、EPSPS探针。M: DNA marker; P: pTGH-1质粒(阳性对照); N: J9707 (阴性对照); 1~9: 转基因阳性植株, 依次为EPS-1的T2代植株、EPS-2的T2代植株、EPS-3的T2代植株、EPS-4的T2代植株、EPS-5的T2代植株、EPS-6的T2代植株、EPS-6的T3代植株、EPS-7的T2代植株、EPS-7的T3代植株。"

表2

单拷贝转基因家系草甘膦抗性分离鉴定"

株系编号
Line ID		 总株数
Total plant number		 存活株数
Number of survived plants		 死亡株数
Number of dead plants		 期望分离比
Expected segregated ratio		 χ20.05, 1
EPS-1 50 33 17 3:1 1.71
EPS-2 50 31 19 3:1 3.84
EPS-4 56 39 17 3:1 0.59
EPS-5 49 34 15 3:1 0.55

图5

EPS-2、EPS-6和EPS-7等油菜转化体中T-DNA插入位置信息"

图6

EPS-2、EPS-6和EPS-7等转基因家系的转化体特异性PCR检测结果 A: EPS-2转基因家系不同世代材料的转化体特异性PCR检测结果, 检测所用引物为EPSPS-2 F和P5; B: EPS-6转基因家系不同世代材料的转化体特异性PCR检测结果, 检测所用引物为EPSPS6 F和P5; C: EPS-7转基因家系不同世代材料的转化体特异性PCR检测结果, 检测所用引物为EPSPS7F和P5。其中, M为2 kb DNA marker; P为pTGH-1质粒(阳性对照); N为转化受体J9707 (阴性对照); 1为T0代植株; 2~4为T1代抗性植株; 5~7为T2代抗性植株; 8~10为T3代抗性植株。"

图7

转I. variabilis EPSPS阳性株系的表达检测 A: RT-PCR检测目的基因在T0代株系叶片中的RNA表达水平。其中, M为3 kb DNA marker; 1~5依次为EPS-1、EPS-6、EPS-7、EPS-2和EPS-5等转化系; N: J9707 (阴性对照); P: pTGH-1质粒(阳性对照)。B: Western blot检测目的基因在T0代株系叶片中的蛋白表达水平。其中, N: J9707 (阴性对照); 1~6依次为EPS-1、EPS-6、EPS-36、EPS-7、EPS-2和EPS-5转化系。C: qRT-PCR检测目的基因在T3代株系的不同组织中相对表达量。油菜Actin2 (AF111812)为内参基因。"

图8

转I. variabilis EPSPS基因T1代株系喷施草甘膦处理下的抗性表现"

图9

转I. variabilis EPSPS基因T3代株系苗期喷施不同浓度草甘膦处理下的抗性表现"

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