Functioal Analysis on GmKTI1-like Gene of Soybean Resistance to Bean Pyralid (Lamprosema indicata)

doi:10.13560/j.cnki.biotech.bull.1985.2024-1136

Abstract

Abstract:

Objective Kunitz-type trypsin inhibitors play an important role in enhancing plant resistance to pests. Prior research conducted by our group has demonstrated a significant association between the Kunitz-type trypsin inhibitor GmKTI1-like and resistance to the soybean leaf borer. This study is aimed to investigate the function of the GmKTI1-like gene and investigate its role to provide key resistance genes and new breeding materials for the cultivation of insect-resistant soybean varieties. Method GmKTI1-like was cloned from soybean leaves. Then bioinformatics methods were used to analyze their physicochemical properties, protein structure, physical positioning of chromosomes and subcellular localization, and RT-qPCR was used to analyze the expression pattern of GmKTI1-like in different soybean tissues. Trangenic plants were created using the Agrobacterium-mediated. Following extensive molecular biology research and phenotypic characterization over serveral years, a new transgenic soybean germplasm was identified. This germplasm, which carries the target genes and exhibits genetic stability, was evaluated for its significant application potential. The evaluation included assessments of its insecticial proper ties and measurements of trypsin inhibitor enzyme activity. Result GmKTI1-like was located on chromosome 1, and its encoded GmKTI1-like contained one KTI structural domain and one transmembrane structural domain, and the results of subcellular localization showed that GmKTI1-like was a membrane protein. Fluorescence quantitative PCR showed that GmKTI1-like had the highest expression in soybean leaves. Compared with the wild type (WT), the transgenic soybean plants showed significantly enhanced resistance to insect and high levels of trypsin inhibitor under Lamprosema indicata borer stress. Conclusion The overexpression of GmKTI1-like transgenic soybean lines significantly increase theresistance to insect, indicating its pivotal role in the soybean defense mechaism against the L. indicata borer.

Key words: soybean, Lamprosema indicata, trypsinprotease inhibitor, GmKTI1-like

TAN Yu-rong, CHEN Dong-liang, YANG Shou-zhen, LAI Zhen-guang, TANG Xiang-min, SUN Zu-dong, ZENG Wei-ying. Functioal Analysis on GmKTI1-like Gene of Soybean Resistance to Bean Pyralid (Lamprosema indicata)[J]. Biotechnology Bulletin, 2025, 41(6): 99-108.

Figures/Tables 10

Table 1 Primers and probes used in this study

用途Usage

引物和探针名称 Primer and probe name

引物序列 Primer sequence （5′‒3′）

普通PCR

CP4-EPSPS-F

CP4-EPSPS-R

CCTTCATGTTCGGCGGTCTCG

GCGTCATGATCGGCTCGATG

GmKTI1-like J-F

GmKTI1-like J-R

ATGAAGAGTACCTTGTTCGCC

CATGCAGATGAAAGAGTTAATCCT

qPCR

CYP2-F

CYP2-R

CGGGACCAGTGTTCTTCTTCA

CCCCTCCACTACAAAGGCTCG

GmKTI1-like Q-F

GmKTI1-like Q-R

TACCTTGTTCGCCCTCTTTCTACTT

GCATATTCTATTCCACCGCCGTTTA

TaqMan qPCR

Lectin-F

Lectin-R

Probe1

CTCTACTCCACCCCCATCCA

GAAGGAAGCGGCGAAGCT

FAM-TGGGACAAAGAAACCGGTAGCGTTGC-TAMRA

Nos-F

Nos-R

Probe2

ACGATTTCAAGCGCATCATC

TCACTATTCACCAGTAACAGCAG

FAM-TATAAGTATCTTCCTGGG-TAMRA

Fig. 1 Amino acid sequence analysis and structure prediction of GmKTI1-likeA: Amino acid sequence analysis. B: Conservative domain. C: Comparison. D: Distribytion of GmKTI1-like gene on soybean chrommosomes. D: Signal peptide. E: Tertiary structure of the protein

Fig. 2 Subcellular location of the GmKTI1-like fusion in epidermis cells of transformed tobacco

Fig. 3 Expression pattern of GmKTI1-like gene in different tissues of soybeanA: Expression pattem of GmKTI1-like in different tissues of soybean based on RNA-seq data. B: RT-qPCR assays of the tissue expression pattern of GmKTI1-like in different tissues of soybean. Different letters in the figure indicate significant differences (P≤0.05). The same below

Fig. 4 Molecular detection of some overexpressed plantsA: Protein test strip assay detection of the exogenous gene CP4-EPSPS. B: PCR detection of the exogenous gene CP4-EPSPS. C: PCR detection of the gene GmKTI1-like; M1: DL 2000 DNA marker; M2: BM2000+ DNA marker

Fig. 5 Expressions of the target gene GmKTI1-like in some transgenic soybean plantsCK indicates non-transgenic line, other lines indicate transgenic line

Fig. 6 Standard curve of the endogenous gene Lectin (A) and the exogenous sequence Nos (B)

Table 2 Copy number of exogenous sequences in transgenic soybean plants of T2 generation

T₁代植株编号 T₁ plant No.	C_tR （Lectin）	C_tX （Nos）	X₀/R₀
GmKTI1-like-4-8	26.66	28.64	1.01
GmKTI1-like-5-9	27.20	28.71	1.05
GmKTI1-like-6-4	26.80	27.22	1.10
GmKTI1-like-6-9	24.72	28.78	0.88
GmKTI1-like-7-2	27.57	28.68	1.08
GmKTI1-like-9-2	26.31	28.71	0.98
GmKTI1-like-12-1	27.79	28.90	1.08
GmKTI1-like-15-6	25.91	28.08	0.99

Fig. 7 Identification of indoor inoculationA: Leaf feeding after 48 h of inoculation (a, b indicate non-transgenic plants, c‒i indicate transgenic plants). B: Weight of remaining leaves after 48 h of inoculation (CK indicates non-transgenic plants, others indicate transgenic plants)

Fig. 8 Enzymatic activities of trypsin inhibitors in GmKTI1-like overexpressed lines subjected to bean leaf roller stress** indicates highly significant differences between strains at the P≤0.01 level. CK indicates non-transgenic plants, others indicate transgenic plants

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