Identification and Resistance Detection of Homozygous and Heterozygous Plants of Transgenic Maize LD05 with Resistances to Insect and Herbicide

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

Abstract

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

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.

Figures/Tables 10

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

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

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

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

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

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

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)

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

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

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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