转基因抗虫耐除草剂玉米LD05纯杂合植株的鉴定及抗性检测

doi:10.13560/j.cnki.biotech.bull.1985.2024-0877

摘要/Abstract

摘要：

目的在回交转育过程中利用分子检测手段将转基因玉米LD05目标性状准确快速导入育种常规自交系中，并明确LD05纯杂合株系和对照郑58在抗虫融合基因m2cryAb-vip3A表达水平、抗虫性和农艺性状等方面是否存在差异。方法通过左右边界引物进行PCR扩增鉴定外源目的基因m2cryAb-vip3A在自交系中的纯杂合，利用RT-qPCR和ELISA开展m2cryAb-vip3A在转录和翻译水平的表达分析，并通过室内生测和田间接虫试验鉴定LD05纯杂合株系对靶标害虫亚洲玉米螟、草地贪夜蛾、黏虫和棉铃虫的抗性，通过田间调查和室内考种对LD05纯杂合株系和对照郑58在农艺性状方面的差异进行比较分析。结果通过筛选优化鉴定，确定LC915+LC966为最优纯杂合鉴定引物。纯杂合株系中外源插入基因m2cryAb-vip3A在转录和翻译水平存在差异，纯合株系中普遍高于杂合株系。室内生测结果显示，喂食LD05纯合株系和杂合株系心叶期叶片，玉米螟、草地贪夜蛾、黏虫的校正死亡率均达到100%，为高抗级别；田间接虫试验结果显示，LD05纯合株系和杂合株系在心叶期和花丝期对玉米螟、心叶期对黏虫、花丝期对棉铃虫的抗性等级均为高抗。农艺性状调查显示LD05纯合株系、杂合株系和对照郑58无差别。结论建立了基于普通PCR的LD05转化体中目的基因的纯杂合鉴定方法，明确外源目的基因m2cryAb-vip3A在LD05纯杂合株系中表达存在差异，但在抗虫性和农艺性状等方面没有显著差异。

关键词: m2cryAb-vip3A基因, 纯杂合鉴定, 抗虫性, 转基因, 玉米

Abstract:

Objective In the process of backcross transfer, the target trait of transgenic maize LD05 was accurately and quickly introduced into the conventional inbred lines of breeding by molecular detection methods, and it was determined whether there were differences in the expressions of the insect-resistant fusion gene m2cryAb-vip3A, insect-resistant and agronomic traits between homozygous and heterozygous individuals of LD05 and the control Zheng 58. Method Homozygous and heterozygous expressions of foreign target gene m2cryAb-vip3A in inbred lines were identified by PCR amplification using left and right boundary primers, and the expressions of m2cryAb-vip3A at transcription and translation levels were analyzed by RT-qPCR and ELISA. The resistances to target pests were evaluated by bioassay and field trials. Result LC915+LC966 was identified as the optimal primers for homozygous and heterozygous identification by screening and optimization. The transcription and translation levels of exogenous gene m2cryAb-vip3A were different in homozygous and heterozygous lines, and those were generally higher in homozygous lines than in heterozygous lines. The results of laboratory bioassay showed that the corrected mortality rates of Ostrinia furnacalis, Spodoptera fragiperda and Mythimna separata were 100% when feeding LD05 homozygous and heterozygous lines at the heart leaf stage, indicating high resistance level. The results of field trials showed that the resistance levels of LD05 homozygous and heterozygous lines to O. furnacalis in the heart leaf stage and silking stage, Mythimna separata in heart leaf stage and Helicoverpa armigera in silking stage were high. Agronomic character investigation showed that there was no difference between the homozygous and heterozygous lines of LD05 and Zheng 58. Conclusion The homozygous and heterozygous identification methods of target genes in LD05 transforms are established based on ordinary PCR. It is confirmed that the expression of the exogenous target gene m2cryAb-vip3A is different in the homozygous and heterozygous lines of LD05, but there is no significant difference in resistance to insect and agronomic traits.

Key words: gene m2cryAb-vip3A, homozygous and heterozygous identification, resistance to insect, transgenic, maize

李文兰, 侯辛未, 李燕, 赵瑞君, 孟昭东, 岳润清. 转基因抗虫耐除草剂玉米LD05纯杂合植株的鉴定及抗性检测[J]. 生物技术通报, 2025, 41(4): 123-133.

LI Wen-lan, HOU Xin-wei, LI Yan, ZHAO Rui-jun, MENG Zhao-dong, YUE Run-qing. Identification and Resistance Detection of Homozygous and Heterozygous Plants of Transgenic Maize LD05 with Resistances to Insect and Herbicide[J]. Biotechnology Bulletin, 2025, 41(4): 123-133.

图/表 10

图1 LD05转化体特异性PCR引物示意图

Fig. 1 Schematic diagram of LD05 transformer-specific PCR primers

表1 设计引物的序列信息

Table 1 Sequence information for designing primers

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'）
LC913	AAGTGTTCAGTAAATTATAT	LC953	TGGCGTTACCCAACTTAATC
LC915	ATACGCCTGTCAAGTGTCAT	LC958	AATTGGTTCTTGGAATCGCA
LC917	CCTTGTCCTGCATTGCGCAT	LC962	ATATGTTTCTCAGCGAGCAT
LC937	TCTACTTGGCAAAGGCTTCAGAT	LC964	CACGGTTCCAGAGGAAAACC
LC947	CAGTAAATTATATACGCCTG	LC966	GTGCTAGTCAATAGAACTA
LC936	GCACCATCGTCAACCACTACA	LC968	CACATCGCTAGCTAGTGCTA
LC938	TCTGGCAGCTGGACTTCAGCCTG	LC928	CGCATAGAAACAACAGAAGTG
LC944	CCAGTACTAAAATCCAGATC	LC929	TCCTAAAACCAAAATCCAGTA
LC948	CGTCCGCAATGTGTTATTAA	LC933	GGCAGAGGCATCTTCAACG
LC931	CTTGATGAGACCTGCTGCGT	LC934	AAACCCCAAGTCCAAGTAAC
LC941	TGAGACCTGCTGCGTAAGCC	zSSIIb-F	CTCCCAATCCTTTGACATCTGC
LC951	AGCTTGGCACTGGCCGTCG	zSSIIb-R	TCGATTTCTCTCTTGGTGACAGG

表2 设计定量引物的序列信息

Table 2 Sequence information of the designed RT-qPCR primers

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'）
m2cryAb-vip3A-F	GTTTCCTTTACCGGGGACGA	18S-F	AAACGGCTACCACATCCAAG
m2cryAb-vip3A-A	ACCACCCCCTTCAACTTCAG	18S-A	CCTCCAATGGATCCTCGTTA

图2 LD05中外源目的基因的纯杂合鉴定引物筛选A：LD05转化体特异性PCR引物筛选；M：DL2000 Maker；1：引物LC937和LC936的扩增产物；2：引物LC937和LC938的扩增产物；3：引物LC937和LC944的扩增产物；4：引物LC937和LC948的扩增产物；5：引物LC947和LC936的扩增产物；6：引物LC947和LC938的扩增产物；7：引物LC947和LC944的扩增产物；8：引物LC947和LC948的扩增产物；9：引物LC917和LC936的扩增产物；10：引物LC917和LC938的扩增产物；11：引物LC917和LC944的扩增产物；12：引物LC917和LC948的扩增产物；13：引物LC913和LC936的扩增产物；14：引物LC913和LC938的扩增产物；15：引物LC913和LC944的扩增产物；16：引物LC913和LC948的扩增产物；17：引物LC915和LC936的扩增产物；18：引物LC915和LC938的扩增产物；19：引物LC915和LC944的扩增产物；20：引物LC915和LC948的扩增产物；21：引物LC928和LC929的扩增产物；22：引物LC931和LC962的扩增产物；23：引物LC941和LC962的扩增产物；24：引物LC951和LC962的扩增产物；25：引物LC953和LC962的扩增产物；26：引物LC931和LC964的扩增产物；27：引物LC941和LC964的扩增产物；28：引物LC951和LC964的扩增产物；29：引物LC953和LC964的扩增产物；30：引物LC931和LC966的扩增产物；31：引物LC941和LC966的扩增产物；32：引物LC951和LC966的扩增产物；33：引物LC953和LC966的扩增产物；34：引物LC931和LC968的扩增产物；35：引物LC941和LC968的扩增产物；36：引物LC951和LC968的扩增产物；37：引物LC953和LC968的扩增产物；38：引物LC931和LC958的扩增产物；38：引物LC941和LC958的扩增产物；40：引物LC951和LC958的扩增产物；41：引物LC953和LC958的扩增产物；42：引物LC933和LC934的扩增产物。B：LD05中外源目的基因的纯杂合鉴定引物筛选；1：纯合DNA样本；2：杂合DNA样本；3：野生型DNA样本；M：DL2000 marker，条带从上往下依次代表2 000、1 000、750、500、250、100 bp

Fig. 2 Screening of primers for homozygous and heterozygous identification of target gene in LD05A: Screening of specific PCR primers for LD05 transformants. M: DL2000 maker. 1: Amplified products of primers LC937 and LC936. 2: Amplified products of primers LC937 and LC938. 3: Amplified products of primers LC937 and LC944. 4: Amplified products of primers LC937 and LC948. 5: Amplified products of primers LC947 and LC936. 6: Amplified products of primers LC947 and LC938. 7: Amplified products of primers LC947 and LC944. 8: Amplified products of primers LC947 and LC948. 9: Amplified products of primers LC917 and LC936. 10: Amplified products of primers LC917 and LC938. 11: Amplified products of primers LC917 and LC944. 12: Amplified products of primers LC917 and LC948. 13: Amplified products of primers LC913 and LC936. 14: Amplified products of primers LC913 and LC938. 15: Amplified products of primers LC913 and LC944. 16: Amplified products of primers LC913 and LC948. 17: Amplified products of primers LC915 and LC936. 18: Amplified products of primers LC915 and LC938. 19: Amplified products of primers LC915 and LC944. 20: Amplified products of primers LC915 and LC948. 21: LC928 and LC929. 22: Amplified products of primers LC931 and LC962. 23: Amplified products of primers LC941 and LC962. 24: Amplified products of primers LC951 and LC962. 25: Amplified products of primers LC953 and LC962. 26: Amplified products of primers LC931 and LC964. 27: Amplified products of primers LC941 and LC964. 28: Amplified products of primers LC951 and LC964. 29: Amplified products of primers LC953 and LC964. 30: Amplified products of primers LC931 and LC966. 31: Amplified products of primers LC941 and LC966. 32: Amplified products of primers LC951 and LC966. 33: Amplified products of primers LC953 and LC966. 34: Amplified products of primers LC931 and LC968. 35: Amplified products of primers LC941 and LC968. 36: Amplified products of primers LC951 and LC968. 37: Amplified products of primers LC953 and LC968. 38: Amplified products of primers LC931 and LC958. 38: Amplified products of primers LC941 and LC958. 40: Amplified products of primers LC951 and LC958. 41: Amplified products of primers LC953 and LC958. 42: Amplified products of primers LC933 and LC934. B: Screening of primers for homozygous and heterozygous identification of target gene in LD05; 1: homozygous DNA sample; 2: heterozygous DNA sample; 3: wild-type DNA sample; M: DL2000 marker, the bands from top to bottom represent 2 000, 1 000, 750, 500, 250, and 100 bp

图3 不同引物组合的扩增循环数对扩增效率的影响和检出限测试A：不同引物组合的扩增循环数对扩增效率的影响；1、7、13、19、25、31：循环数20；2、8、14、20、26、32：循环数22；3、9、15、21、27、33：循环数24；4、10、16、22、28、34：循环数26；5、11、17、23、29、35：循环数28；6、12、18、24、30、36：循环数30；M：DL2000 marker，条带从上往下依次代表2 000、1 000、750、500、250、100 bp。B：不同引物组合的检出限测试；1、7、13、19、25、31：模板浓度为100 ng/µL；2、8、14、20、26、32：模板浓度为10 ng/µL；3、9、15、21、27、33：模板浓度为1 ng/µL；4、10、16、22、28、34：模板浓度为0.1 ng/µL；5、11、17、23、29、35：模板浓度为0.01 ng/µL；6、12、18、24、30、36：模板浓度为0.001 ng/µL；M：DL2000 marker

Fig. 3 Effects of different amplification cycles on amplification efficiency and detection limit test of different primer combinationsA: Effect of different amplification cycles on amplification efficiency; 1, 7, 13, 19, 25, 31: 20 cycles; 2, 8, 14, 20, 26, 32: 22 cycles; 3, 9, 15, 21, 27, 33: 24 cycles; 4, 10, 16, 22, 28, 34: 26 cycles; 5, 11, 17, 23, 29, 35: 28 cycles; 6, 12, 18, 24, 30, 36: 30 cycles; M: DL2000 marker, the bands from top to bottom represent 2 000, 1 000, 750, 500, 250, 100 bp. B: Detection limit test of different primer combinations; 1, 7, 13, 19, 25, 31: template concentration is 100 ng/µL; 2, 8, 14, 20, 26, 32: template concentration of 10 ng/µL; 3, 9, 15, 21, 27, 33: the template concentration is 1 ng/µL; 4, 10, 16, 22, 28, 34: the template concentration is 0.1 ng/µL; 5, 11, 17, 23, 29, 35: the template concentration is 0.01 ng/µL; 6, 12, 18, 24, 30, 36: template concentration is 0.001 ng/µL; M: DL2000 marker

图4 纯杂合引物测试分析A：引物LC915+LC966的PCR扩增结果；B：引物LC915+LC948的PCR扩增结果。1-93：BC6F2植株的93个随机DNA样本；94：空白对照；95：阴性对照；96：阳性对照；M：DL2000 marker，条带从上往下依次代表2 000、1 000、750、500、250、100 bp

Fig. 4 Analysis of homozygous and heterozygous primersA: PCR amplified results of primer LC915+LC966; B: PCR amplified results of primer LC915+LC948. 1-93: 93 random DNA samples from BC6F2 plants; 94: blank control; 95: negative control; 96: positive control; M: DL2000 marker, the bands from top to bottom represent 2 000, 1 000, 750, 500, 250, and 100 bp

图5 m2cryAb-vip3A在纯杂合株系中的RT-qPCR和ELISA分析A：m2cryAb-vip3A在纯杂合株系中的RT-qPCR分析；B：m2cryAb-vip3A编码蛋白在纯杂合株系中的ELISA分析。1、5、8、20：杂合株系；3、7、17：纯合株系。柱形条上的字母（或字母数字组合）代表利用邓肯多重检测分析表达差异显著性（P<0.05）

Fig. 5 RT-qPCR and ELISA analysis of m2cryAb-vip3A in homozygous and heterozygous plantsA: RT-qPCR analysis of m2cryAb-vip3A in homozygous and heterozygous plants; B: ELISA analysis of m2cryAb-vip3A encoded protein in homozygous and heterozygous plants. 1, 5, 8, 20: heterozygous plants; 3, 7, 17: homozygous plants. The letters (or alphanumeric combinations) in bars indicate the significant difference in expression analysis by Duncan multiple detection(P<0.05)

图6 玉米心叶期叶片对玉米螟、草地贪夜蛾和黏虫的室内生测结果（接虫后6 d）A-C：郑58心叶期叶片；D-F：纯合植株心叶期叶片；G-I：杂合植株心叶期叶片。A、D、G：试虫为玉米螟；B、E、H：试虫为草地贪夜蛾；C、F、I：试虫为黏虫；J：玉米螟校正死亡率；K：草地贪夜蛾校正死亡率；L：黏虫校正死亡率

Fig. 6 Results of laboratory bioassay on Ostrinia furnacalis, Spodoptera fragiperda and Mythimna separata at the heart leaf stage (6 d after inoculation)A-C: Zheng 58 leaves at V5 stage; D-F: leaves of homozygous plants at V5 stage; G-I: leaves of heterozygous plants atV5 stage.A, D, G: Ostrinia furnacalis; B, E, H: Spodoptera fragiperda; C, F, I: Mythimna separata; J: corrected mortality of Ostrinia furnacalis; K: corrected mortality of Spodoptera fragiperda; L: corrected mortality of Mythimna separata

表3 玉米螟、黏虫和棉铃虫田间接虫结果分析

Table 3 Analysis field trials of Ostrinia furnacalis, Helicoverpa armigera and Helicoverpa armigera

靶标害虫

Target pests

接种时期

Inoculation period

纯合植株

Homozygous plant

杂合植株

Heterozygous plant

郑58

Zheng 58

级值

Level value

抗性

Resistance

级值

Level value

抗性

Resistance

级值

Level value

抗性

Resistance

玉米螟

Ostrinia furnacalis

心叶期

V5 stage

1.0

高抗

High resistance

1.0

高抗

High resistance

8.90

高感

High sense

花丝期

Silking stage

1.0

高抗

High resistance

1.0

高抗

High resistance

8.99

高感

High sense

黏虫

Mythimna separata

心叶期

V5 stage

1.01

高抗

High resistance

1.02

高抗

High resistance

8.65

高感

High sense

棉铃虫

Helicoverpa armigera

花丝期

Silking stage

1.0

高抗

High resistance

1.0

高抗

High resistance

6.85

感

Sense

表4 LD05纯合株系、杂合株系和对照郑58农艺性状比较分析

Table 4 Comparative analysis of agronomic characters between LD05 of homozygous and heterozygous plants and Zheng 58

农艺性状 Agronomic trait	LD05纯合株系 Homozygous plant	杂合株系 Heterozygous plant	郑58 Zheng 58
抽雄期Tasseling stage/d	59.7±1.2a	59.0±1.0a	59.3±0.6a
生育期Growth stage/d	109.0±1.0a	109.3±0.6a	109.7±1.2a
株高Plant height/cm	163.7±1.5a	163.0±4.0a	164.3±1.5a
穗位高Ear height/cm	56.3±2.1a	56.7±0.6a	56.0±1.0a
穗行数Rows per ear	12±0a	12±0a	12±0a
穗粗Ear diameter/cm	3.8±0.1a	3.8±0.0a	3.8±0.1a
百粒重100-kernel weight/g	33.2±0.9a	33.0±0.2a	33.6±0.7a

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