大豆抗豆卷叶螟GmKTI1-like的功能研究

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

摘要/Abstract

摘要：

目的 Kunitz型胰蛋白酶抑制剂在增强植物抵抗害虫方面发挥重要作用。前期研究表明胰蛋白酶抑制剂GmKTI1-like与大豆抗豆卷叶螟密切相关，探究GmKTI1-like基因在豆卷叶螟胁迫中的功能，为培育抗虫新品种提供重要抗性基因和育种新材料。方法从大豆叶片中克隆GmKTI1-like，利用生物信息学方法对其理化性质、蛋白质结构、染色体物理定位、亚细胞定位进行分析，采用RT-qPCR分析不同组织中GmKTI1-like的表达模式；利用农杆菌介导法获得转基因植株，经过多年的分子生物学和表型鉴定，筛选携带目标基因且遗传稳定的转基因抗虫大豆新种质，对其进行接虫鉴定及胰蛋白酶抑制剂酶活测定。结果 GmKTI1-like位于第1染色体上，其编码的GmKTI1-like包含1个KTI结构域和1个跨膜结构域，亚细胞定位分析表明，GmKTI1-like定位于细胞膜上；荧光定量PCR显示，GmKTI1-like在大豆叶片中表达量最高；与野生型（WT）相比，豆卷叶螟胁迫下，转基因大豆植株抗虫性显著增强、胰蛋白酶抑制剂含量高。结论过表达大豆GmKTI1-like转基因株系抗虫性能力显著提高，说明其在大豆抗豆卷叶螟的防御反应中发挥重要作用。

关键词: 大豆, 豆卷叶螟, 胰蛋白酶抑制剂, GmKTI1-like

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

谭玉荣, 陈东亮, 杨守臻, 赖振光, 唐向民, 孙祖东, 曾维英. 大豆抗豆卷叶螟GmKTI1-like的功能研究[J]. 生物技术通报, 2025, 41(6): 99-108.

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.

图/表 10

表1 本研究所用引物及探针

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

图1 GmKTI1-like的氨基酸序列分析与结构预测A：GmKTI1-like氨基酸序列分析；B：保守结构域；C：大豆GmKTI1-like与其他KTI1-like的氨基酸序列比对；D：GmKTI1-like基因在大豆染色体中的定位；E：三级结构；F：信号肽预测

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

图2 转化烟草表皮细胞的GmKTI1-like亚细胞定位

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

图3 GmKTI1-like基因在大豆不同组织中的表达模式A：转录组测序数据分析GmKTI1-like在不同组织的表达；B： RT-qPCR验证GmKTI1-like的组织表达模式。不同小写字母表示不同组织器官间差异显著（P≤0.05）。下同

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

图4 部分过表达植株的分子检测A：外源基因CP4-EPSPS的蛋白试纸条检测；B：外源基因CP4-EPSPS的PCR检测；C：GmKTI1-like的PCR检测；M1： DL 2000 DNA marker；M2： BM 2000+ DNA marker

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

图5 部分转基因大豆植株目标基因GmKTI1-like的表达水平CK为非转基因株系，其他为转基因株系

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

图6 内参基因Lectin（A）和外源序列Nos（B）的标准曲线

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

表2 T2代转基因大豆植株外源序列拷贝数

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

图7 室内接虫鉴定A：接虫48 h后叶片啃食情况（a，b为非转基因植株，c‒i为转基因植株）；B：接虫48 h后剩余叶片重量（CK为非转基因植株，其他为转基因植株）

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)

图8 GmKTI1-like过表达株系受豆卷叶螟胁迫后胰蛋白酶抑制剂的酶活**表示不同株系间P≤0.01水平下存在极显著差异；WT为非转基因植株，其他为转基因植株

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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