杜仲BGL基因创制高效嫁接砧木

doi:10.13560/j.cnki.biotech.bull.1985.2025-1002

摘要/Abstract

摘要：

目的 β-1,4-葡聚糖酶（β-1,4-Glucanase, BGL）是一种可水解细胞壁纤维素的酶，能够通过促进砧木与接穗的愈合有效提高嫁接成活率。解析杜仲（Eucommia ulmoides）BGL基因功能，对建立高效嫁接技术体系及深入开展相关基因功能研究具有重要意义。方法分别构建由创伤诱导型启动子FAR6和生长素诱导型启动子GH3驱动的BGL基因植物表达载体，并通过遗传转化杜仲获得转基因植株。利用微嫁接技术比较不同接穗和砧木组合的成活率，采用RT-qPCR分析伤口愈合相关基因的表达，并通过解剖学观察评估嫁接部位愈伤组织形成和愈合情况。结果 RT-qPCR结果表明，转FAR6::BGL植株中BGL基因受创伤诱导表达，而转GH3::BGL植株中BGL基因受生长素诱导表达。微嫁接实验显示，转FAR6::BGL接穗的成活率（63.33%）显著高于转GH3::BGL接穗（53.33%）和野生型接穗（33.33%）。以转FAR6::BGL植株为砧木时，与野生型接穗的嫁接成活率达80%，显著高于野生型砧木（30%）。此外，转FAR6::BGL砧木可促进伤口愈合相关基因（EuERF115-like、EuNAC1和EuWOX13-2）的表达。解剖学观察表明，其嫁接部位愈伤组织形成更快，伤口愈合时间更短。结论转FAR6::BGL杜仲砧木能显著提高杜仲微嫁接成活率，促进伤口愈合相关基因的表达，加速嫁接部位愈合过程，为克服转基因植株生根困难及移栽成活率低等问题提供新策略。

关键词: 杜仲, β-1,4-葡聚糖酶基因, 砧木, 微嫁接, 成活率, 接穗, 启动子

Abstract:

Objective β-1,4-Glucanase (BGL) is an enzyme that hydrolyzes cellulose in the cell wall and effectively improves graft survival by promoting the healing between rootstock and scion. Elucidating the function of the BGL gene in Eucommiaulmoides is important for establishing an efficient grafting system and further investigating related gene functions. Method Plant expression vectors for the BGL gene, driven by the trauma-inducible promoter FAR6 and the auxin-inducible promoter GH3, were constructed, and transgenic plants were obtained through genetic transformation of E. ulmoides. Micrografting techniques were employed to compare survival rates among different scion-rootstock combinations; RT-qPCR was used to analyze the expression of wound-healing-related genes; and anatomical observations were conducted to evaluate callus formation and healing at the graft union. Result RT-qPCR results indicated that BGL expression was induced by wounding in transgenic FAR6::BGL plants and by auxin in GH3::BGL plants. Micrografting showed that the survival rate of FAR6::BGL scions (63.33%) was significantly higher than that of GH3::BGL (53.33%) and wild-type (WT) scions (33.33%). When FAR6::BGL plants were used as rootstocks grafted with WT scions, the survival rate reached 80%, significantly higher than that with WT rootstocks (30%). Furthermore, FAR6::BGL rootstocks promoted the expression of wound-healing-related genes (EuERF115-like, EuNAC1, and EuWOX13-2). Anatomical observations revealed more rapid callus formation and shorter wound-healing time at the graft junction. Conclusion Transgenic E. ulmoides rootstocks expressing FAR6::BGL significantly enhance micrografting survival rates, promote the expressions of wound-healing-related genes, and accelerate the graft union healing process, thereby providing a novel strategy to overcome challenges such as difficult rooting and low transplant survival in transgenic plants.

Key words: Eucommia ulmoides, β-1,4-glucanase gene, rootstock, micrografting, survival rate, scion, promoter

张珊, 孙志鹏, 赵德刚, 赵懿琛. 杜仲BGL基因创制高效嫁接砧木[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1002.

ZHANG Shan, SUN Zhi-peng, ZHAO De-gang, ZHAO Yi-chen. Eucommia ulmoides BGL Gene for Developing Highly Efficient Grafting Rootstocks[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1002.

图/表 8

表1 引物序列

Table 1 Primer sequences

引物名称 Primer name	引物序列 Primer sequence （5′-3′）
GUS-F	GGTGATTGATGAAACTGCTG
GUS-R	GAACATTACATTGACGCAGG
β-1,4--F	ATGGGTTCTCCACTTACACAGTC
β-1,4--R	TTAAGGTGCTAAATGGATATTCTTG
EuActin-F	GTGTTATGGTTGGGATGGG
EuActin-R	TGCTGACTATGCCGTGTTC
β-1,4-qRT-F	GGTTTGGAGACCACTACCCG
β-1,4-qRT-R	GTGTGTTTGGGTTGGGTTCG
EuERF115-like-qRT-F	CATCGTCATCGTCATCATC
EuERF115-like-qRT-R	ACTAAGGTGGTCCATTGAG
EuNAC1-qRT-F	AAGGATCAGTTGGAGCTGCC
EuNAC1-qRT-R	AGACATTCCTGGAAGCTGCC
EuWOX13-2-qRT-F	GGTCTGAGGGCATGTGTTTT
EuWOX13-2-qRT-R	TTGGAGATATGGGTGGTGGT

图1 转基因杜仲抗性芽的获得与鉴定A：杜仲遗传转化流程图（a：悬浮培养；b：共培养；c：筛选培养；d：诱导愈伤；e：抗性芽）；B：转基因杜仲GUS检测（WT：野生型杜仲；a：转FAR6::BGL杜仲；b：转GH3::BGL杜仲）；C：转基因杜仲PCR扩增鉴定（GUS基因：304 bp），M：DNA DL 2 000 marker；1-12：转FAR6::BGL植株；13-24：转GH3::BGL植株

Fig. 1 Acquisition and identification of transgenic E. ulmoides resistant budsA: E. ulmoides genetic transformation flow chart (a: Suspension culture. b: Co-culture. c: Screening culture. d: Induced callus. e: Resistant bud). B: GUS detection of transgenic E. ulmoides (WT: Wild type E. ulmoides. a: FAR6::BGLE. ulmoides. b: GH3::BGLE. ulmoides). C: PCR amplification of transgenic E. ulmoides (GUS gene:304 bp). M: DNA DL2 000 marker. 1-12: Transfer FAR6::BGL plants. 13-24: Transfer GH3::BGL plants

图2 杜仲中BGL基因的相对表达量分析A：创伤处理；B：10 μmol/L IAA处理；不同小写字母表示在P<0.05水平差异显著。下同

Fig. 2 Analysis of the relative expression of BGL gene in E. ulmoidesA: Injury treatment; B:10 μmol/L IAA treatment. Different lower letters indicate significant differences at P<0.05 level. The same below

图3 嫁接成活率统计（A）及BGL基因表达量分析（B）WT：野生型杜仲；FAR6：转FAR6::BGL杜仲；GH3：转GH3::BGL杜仲。下同

Fig. 3 Statistics of graft survival rate (A) and analysis of BGL gene expression (B)WT: Wild type E. ulmoides; FAR6: FAR6::BGLE. ulmoides; GH3: GH3::BGLE. ulmoides. The same below

图4 转基因杜仲砧木的获得与鉴定A：农杆菌注射法遗传转化杜仲流程图（a：杜仲幼苗；b：拔除胚芽的杜仲幼苗；c：对杜仲幼苗注射菌液；d：转化15 d后的杜仲幼苗；e：转化2个月的杜仲幼苗）；B：转基因杜仲GUS检测（WT：野生型杜仲；TP：转FAR6::BGL杜仲）；C：转基因杜仲PCR扩增鉴定（GUS基因：304 bp），M：DNA DL 2 000 marker；1-10：转FAR6::BGL植株

Fig. 4 Acquisition and identification of transgenic E. ulmoides rootstocksA: Flow chart of genetic transformation of E. ulmoides by Agrobacterium injection (a: E. ulmoides seedlings. b: Remove the germ of E. ulmoides seedlings. c: Inject bacteria solution into E. ulmoides seedlings. d: E. ulmoides seedlings after 15 d of transformation. e: Converted E. ulmoides seedlings for two months). B: GUS detection of transgenic E. ulmoides (WT: Wild type E. ulmoides. TP: FAR6::BGLE. ulmoides). C: PCR amplification of transgenic E. ulmoides (GUS gene:304 bp) M: DNA DL 2 000 marker. 1-10: Transferring FAR6::BGL plant

图5 转基因杜仲砧木接穗中创伤愈合相关基因表达量分析

Fig. 5 Expression analysis of wound healing related genes in the scions of transgenic E. ulmoides rootstock

图6 转基因杜仲作砧木嫁接愈合过程解剖学（A）和外观形态学（B）观察St：砧木；Sc：接穗；IL：隔离层；Ci：愈伤组织；Ca：形成层；V：维管射线

Fig. 6 Observation of the anatomy (A) and appearance and morphology (B) of the grafting and healing process of transgenic E. ulmoides rootstockSt: Rootstock. Sc: Scion. IL: Isolation layer. Ci: Callus. Ca: Cambium. V: Vascular

表2 不同杜仲砧木的微嫁接成活率

Table 2 Survival rate of E. ulmoides micrografting of different rootstocks

砧木类别

Rootstock type

嫁接株数

Number of grafis （plants）

存活株数

Number of survivors （plants）

成活率

Survival rate （%）

野生型杜仲

WT E. ulmoides

转FAR6::BGL杜仲

FAR6::BGL E. ulmoides

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