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基于转基因日本落叶松愈伤组织评价外源植物凝集素基因GNA和ConA的抗松材线虫功能

Evaluation of the Nematicidal Activity of Exogenous Plant Lectin Genes GNA and ConA Against Bursaphelenchus xylophilus Based on Transgenic Larix kaempferi Callus

    摘要
  • 摘要:
    目的 探究植物凝集素基因的抗松材线虫功能，为创制抗松材线虫病的新种质提供候选基因。
    方法 根据日本落叶松密码子的偏好性，对雪花莲凝集素基因（Galanthus nivalis agglutinin，GNA）和刀豆凝集素基因（Canavalia ensiformis agglutinin，ConA）进行改造，分别构建了日本落叶松启动子pLaUBQ驱动的2个植物过表达载体。利用农杆菌介导法转化日本落叶松胚性愈伤组织，通过分子鉴定获得转GNA基因和ConA基因的愈伤组织。接种松材线虫5 d后，从愈伤组织形态、细胞活力、显微结构和线虫繁殖量4个方面，探讨2种转基因愈伤对松材线虫的抗性。
    结果 通过农杆菌介导法将外源基因GNA和ConA分别转入日本落叶松胚性愈伤组织，各自获得21个转基因细胞系。根据基因的表达水平，分别选取表达量较高的5个系（转GNA系：G7、G16、G19、G20、G21；转ConA系：C4、C14、C16、C18、C22）进行抗性检测。结果表明，接种松材线虫5 d后，转基因愈伤组织褐化与水渍化程度显著低于未转化愈伤组织，其中G21与C4未出现明显的损伤；转基因愈伤组织的活力普遍高于未转化愈伤组织，其中G21与C4活力最强；细胞系G21与C4中原胚团结构相对完整，而未转化愈伤组织中原胚团结构被破坏；转基因愈伤组织中线虫数量均显著低于对照（13 167 ± 669条）（p < 0.05），其中G21中线虫数量为8 083 ± 796条，降低38.6%，C4中为6 583 ± 193条，降低50.0%。
    结论 本研究表明，植物凝集素基因GNA和ConA具有抗松材线虫功能，可以用来创制抗松材线虫病的新种质。本研究不仅构建了日本落叶松转基因愈伤组织的松材线虫抗性评价体系，也为转基因林木抗虫性的早期鉴定提供了有力工具。

     

    Abstract:
    Objective To investigate the function of plant lectin genes against Bursaphelenchus xylophilus (pine wood nematode) and provide candidate genes for developing new germplasm resistant to pine wilt disease (PWD).
    Methods Based on the codon preference of Larix kaempferi, the Galanthus nivalis agglutinin (GNA) gene and Canavalia ensiformis agglutinin (ConA) gene were modified. Two plant overexpression vectors were constructed, each driven by the L. kaempferi promoter pLaUBQ. The L. kaempferi embryogenic callus were transformed via Agrobacterium-mediated method. Transgenic callus carrying GNA gene and ConA gene were obtained through molecular identification. Five days after inoculation with B. xylophilus, resistance was evaluated based on callus morphology, cell viability, microscopic structure, and nematode reproduction.
    Results TBoth GNA and ConA were successfully integrated into L. kaempferi embryogenic callus, generating 21 transgenic cell lines for each gene. Five high-expression lines per gene (GNA: G7, G16, G19, G20, G21; ConA: C4, C14, C16, C18, C22) were used for resistance assessment. After five days post-inoculation, transgenic callus exhibited less browning and water-soaking compared with untransformed callus, with G21 and C4 showing no visible damage. Cell viability was significantly higher in transgenic lines than untransformed callus, with G21 and C4 displaying the strongest viability. Proembryonic masse remained relatively intact in lines G21 and C4, while those of untransformed callus were disrupted. Nematode number in all transgenic callus were significantly lower than that in untransformed callus (13 167 ± 669 nematodes) (P < 0.05). Line G21 contained 8 083 ± 796 nematodes (38.6% reduction), and line C4 contained 6 583 ± 193 nematodes (50.0% reduction).
    Conclusion This findings demonstrate that GNA and ConA confer resistance to B. xylophilus, and constitute promising candidate genes for developing new PWD-resistant germplasm. Furthermore, this study establishes an evaluation system for assessing the resistance of L. kaempferi transgenic callus to B. xylophilus and offers an effective tool for early evaluation of PWD resistance in transgenic forest trees.

     

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