调控棘孢木霉几丁质酶Tachi2基因的转录因子和蛋白的互作研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0992

摘要/Abstract

摘要：

目的生防菌棘孢木霉（Trichoderma asperellum）TD3104产生的几丁质酶Tachi2在植物病害防治过程中发挥重要作用，47号转录因子作用于特异响应几丁质诱导的Tachi2基因启动子顺式作用元件。探究47号转录因子与一种新调控蛋白H63的互作关系，为解析几丁质诱导调控基因转录表达机制提供科学依据。方法利用酵母双杂交系统对棘孢木霉几丁质酶基因Tachi2中47号转录因子的候选互作蛋白H63进行体内点对点互作鉴定；采用大肠杆菌表达系统分别对H63蛋白与47号转录因子基因进行原核诱导表达，利用亲和层析技术纯化融合蛋白，通过GST pull-down实验进行体外蛋白互作检测；利用农杆菌介导的洋葱表皮细胞亚细胞定位技术和BiFC实验进一步检测细胞内蛋白的相互作用。结果 H63蛋白与47号转录因子在酵母细胞内存在互作关系；原核表达的重组H63蛋白、47号转录因子大小分别为36 kD和18 kD，二者在体外存在互作关系；成功构建了双分子荧光互补载体，证实H63蛋白与47号转录因子在洋葱内表皮细胞中存在相互作用，并且互作发生在细胞核内。结论证实H63蛋白与47号转录因子在细胞内外均存在相互作用，为解析真菌几丁质酶基因的表达调控、几丁质酶在农业和生物医药领域的开发利用奠定理论基础。

关键词: 棘孢木霉, 几丁质酶, 转录因子, 基因表达, 互作蛋白, 酵母双杂交, GST Pull-down, 双分子荧光互补

Abstract:

Objective The chitinase Tachi2, produced by the biocontrol fungus Trichoderma asperellum TD3104, is recognized as playing a significant role in the process of plant disease control. The transcription factor 47 is known to act on the specific response to chitin-induced Tachi2 gene promoter cis-acting elements. The clarification of the interaction between transcription factor 47 and a novel regulatory protein H63 is sought, providing a scientific basis for elucidating the transcriptional expression mechanism of chitin induced regulatory genes. Method The yeast two-hybrid system was employed to identify the candidate interacting protein H63 of transcription factor 47 within the chitinase gene Tachi2 of T. asperellumin vivo. Prokaryotic induction expression of H63 protein and transcription factor 47 gene was performed using an Escherichia coli expression system. The fusion protein was purified using affinity chromatography technology, and protein interactions were detected in vitro using GST pull-down experiments. Further verification of the interaction relationship was conducted through Agrobacterium mediated subcellular localization technique and BiFC experiment of onion epidermal cells. Result It was observed that the H63 protein interacts with transcription factor 47 within yeast cells. The recombinant H63 protein and transcription factor 47 expressed in prokaryotic cells have sizes of 36 kD and 18 kD, respectively, and there is an interaction between the two in vitro. A bimolecular fluorescent complementary vector was successfully constructed, the interaction between H63 protein and transcription factor 47 in onion epidermal cells was confirmed, and the interaction occurred in the nucleus. Conclusion This confirms the interaction between H63 protein and transcription factor 47 both inside and outside the cell, laying a theoretical foundation for analyzing the expression regulation of fungal chitinase genes and the development and utilization of chitinase in agriculture and biomedicine.

Key words: Trichoderma asperellum, chitinase, transcription factor, gene expression, interacting proteins, yeast two hybrid, GST pull-down, BiFC

曲珊, 赵月, 李雅华, 郑桂玲, 咸洪泉. 调控棘孢木霉几丁质酶Tachi2基因的转录因子和蛋白的互作研究[J]. 生物技术通报, 2025, 41(5): 310-319.

QU Shan, ZHAO Yue, LI Ya-hua, ZHENG Gui-ling, XIAN Hong-quan. A Study on the Interaction between Transcriptional Factor and Protein of Tachi2 Chitinase Gene in Trichoderma asperellum[J]. Biotechnology Bulletin, 2025, 41(5): 310-319.

图/表 11

表1 实验所用引物

Table 1 Primers used in the experiment

引物名称 Primer name	序列 Sequence （5′-3′）	限制性内切酶 Restriction enzyme	用途 Usage
47S	CCGGAATTCATGCCGGATGCGGTTACAGAG	EcoR Ⅰ	点对点验证
47A	CGGGATCCTCAAGGCCCACACACCTCCGA	BamH Ⅰ	点对点验证
H63S	TCCCCCCGGGATGGGGGTCCGGCGGC	Xma I	点对点验证
H63A	CGGGATCCTACGGATGCGAAAAGTTGGT	BamH Ⅰ	点对点验证
5′AD	CTATTCGATGATGAAGATACCCCACCAAACCC	-	点对点验证
3′AD	AGTGAACTTGGGGGGTTTTTCAGTATCTACGAT	-	点对点验证
H63S-B/N	CGGGATCCATGGGGTCCGGCGGC	BamH Ⅰ	GST pull-down
H63A-B/N	ATTTGCGGCCGCCTACGGATGCGAAAAGTTGGT	Not I	GST pull-down
CE63S	CGGGATCCATGGGGGTCCGGCGGC	BamH I	BiFC
CE63A	GGGGTACCATCGAAAATATGACCGCACT	Kpn I	BiFC
NE47S	CGGGATCCATGCCGGATGCGGTTACAGAG	BamH I	BiFC
NE47A	GGGGTACCAGGCCCACACACCTCCGA	Kpn I	BiFC

表2 GST pull-down实验设计

Table 2 GST pull-down experimental design

组别 Group	蛋白 Protein
实验组1	GST-H63蛋白+His-47蛋白
实验组2	GST标签蛋白+His-47蛋白
实验组3	GST标签蛋白
对照组1	纯化的His-47蛋白
对照组2	纯化的GST-H63蛋白

图1 pGBKT7-47载体构建A：棘孢木霉RNA；B：PGBKT7-47双酶切验证（1：pGBKT7-47双酶切产物；M：DL5000 bp DNA marker）

Fig. 1 pGBKT7-47 vector constructionA: RNA of Trichoderma asperellum; B: PGBKT7-47 double enzyme digestion verification (pGBKT7-47 double enzyme-digested product; M: 5000 bp DNA marker)

图2 pGADT7-47 载体自激活和毒性检测A：pGBKT7-47/pGADT7的Y2H Gold酵母转化子；B：pGADT7-T/pGBKT7-lam的Y2H Gold酵母转化子；C：pGADT7-T/pGBKT7-53的Y2H Gold酵母转化子

Fig. 2 pGADT7-47 vector self-activation and toxicity testA: Y2H Gold yeast transformant of pGBKT7-47 and pGADT7; B: Y2H Gold yeast transformant of pGADT7-T and pGBKT7-lam; C: Y2H Gold yeast transformant of pGADT7-T and pGBKT7-53

图3 pGADT7-63载体构建A：H63基因PCR产物；B：H63菌液PCR产物验证；C：pGADT7-63双酶切产物；M：DL5000 bp DNA marker

Fig. 3 Construction of interacting pGADT7-H63 vectorA: H63 gene PCR product; B: H63 bacterial liquid PCR product validation; C: PGADT7-63 double enzyme digestion product; M: DL5000 bp DNA marker

图4 酵母双杂交验证pGBKT7-47与 pGADT7-H63互作A：pGBKT7-47/pGADT7-H63的Y2H Gold酵母转化子；B：pGADT7-T/pGBKT7-53的Y2H Gold酵母转化子

Fig. 4 Verification of the interaction of pGBKT7-47 with pGADT7-H63 through yeast two-hybridA: Y2H Gold yeast transformant of pGBKT7-47 and pGADT7-H63; B: Y2H Gold yeast transformant of pGADT7-T and pGBKT7-53

图5 pGEX4T-H63载体构建A：H63基因PCR产物；B：pGEX4T-H63双酶切产物；M：DL5000 bp DNA marker

Fig. 5 Construction of pGEX4T-H63 vectorA: H63 gene PCR product; B: pGEX4T-H63 double enzyme digestion product; M: DL5000 bp DNA marker

图6 重组蛋白SDS-PAGE电泳A：纯化后的47蛋白；B：纯化后的GST-H63蛋白；C：纯化后的GST蛋白；M ：蛋白分子量标准

Fig. 6 SDS-PAGE electrophoresis map of recombinant proteinA: Purified 47 protein; B: purified pGEX4T-1-H63 protein; C: purified pGEX4T-1 protein; M: standard protein marker

图7 GST-H63和His-47 蛋白体外互作验证1：GST-H63和His-47蛋白；2：His-47 和GST蛋白；3：GST蛋白；4：His-47蛋白；5：GST-H63蛋白；M：蛋白分子量标准

Fig. 7 Protein interaction validation of GST-H63 and His-47 in vitro1: GST-H63 protein and His-47 protein; 2: His-47 protein and GST protein; 3: GST; 4: His-47 protein; 5: GST-H63 protein; M: standard protein marker

图8 BiFC实验载体构建A：H63基因PCR产物；B：47号转录因子基因PCR产物；C：菌液PCR验证（1：DH5α/B-T1-47菌液PCR产物；2：DH5α/B-T1-H63菌液PCR产物）；D：双酶切验证（1：NE-47双酶切产物；2：CE-63双酶切产物）；M ：DL5000 bp DNA marker

Fig. 8 Construction of BiFC experimental carrierA: H63 gene PCR product; B: PCR product of transcription factor gene 47; C: PCR validation of bacterial liquid (1: PCR product of DH5 α/B-T1-47 bacterial liquid; 2: PCR product of DH5 α/B-T1-H63 bacterial liquid); D: double enzyme digestion validation (1: NE-47 double enzyme cleavage product; 2: CE-63 double enzyme cleavage product); M: DL5000bp DNA marker

图9 BiFC实验验证蛋白互作A: GV3101/NE-47 and GV3101/CE-H63; B: GV3101/NE and GV3101/CE; C: GV3101/NE and GV3101/CE-H63; D: GV3101/CE and GV3101/NE-47

Fig. 9 BiFC experiment validating protein interaction

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