晶胞粘连苏云金芽胞杆菌C15杀线虫毒力因子发掘与功能鉴定

苏云金芽胞杆菌（Bacillus thuringiensis,Bt）制剂作为一种高效的微生物杀虫剂,在植物病虫害防控领域有着广泛的应用。Bt制剂的主效成分为杀虫晶体和芽胞,其中,杀虫晶体的环境低持久性是Bt农药应用的重要限制因素之一。自然界中存在着一些Bt菌株,其产生的杀虫晶体位于芽胞外壁和芽胞衣之间,这种特殊的表型被称为晶胞粘连（spore-crystal association, SCA）表型。由于芽胞外壁对晶体的保护作用,SCA表型可以提升晶体抵抗不良环境因素的能力,是开发新型Bt生物囊杀虫剂的有效育种策略。本文选取对线虫具有强毒杀能力的 SCA菌株C15作为研究对象。获得了C15菌株的完整基因组序列,包括一个5 637 049 bp的环状染色体和8个不同大小的环形质粒（240 314 bp到3 188 bp）。C15基因编码了5个杀虫蛋白（Cry蛋白）基因：cry21-99、cry21-67、cry21-66、cry21-46和cry-N。在Bt无晶体突变株BMB171中异源表达cry21-99基因,发现其表达产物形成菱形晶体,且对秀丽隐杆线虫（Caenorhabditis elegans）和南方根结线虫（Meloidogyne incognita）均有毒杀活性。同时,还在全基因组范围内预测了Cry毒素以外的杀线虫毒力因子和次生代谢产物。此外,在C15基因组中预测了本团队已报道的苏云金芽胞杆菌幕虫亚种（B. thuringiensis serovar finitimus）菌株YBT-020 SCA表型决定因子的同源基因,缺失后突变体仍然保留稳定的SCA表型,说明C15菌株的SCA表型形成机制与YBT-020不同,该菌株代表了一种新的SCA表型形成机制。本研究为转基因作物防控线虫提供了新的遗传资源,也为研究SCA表型形成机制,开发新型高效Bt制剂提供了新线索。

Identification and characterization of nematocidal factors in Bacillus thuringiensis spore crystal association (SCA) phenotype strain C15

As one of the most successful microbial pesticide, Bacillus thuringiensis (Bt) products are widely used in the field of plant disease and pest control. The main components of Bt products are insecticidal crystals and spores. One of the limiting factors in Bt based products usage in agriculture is that the sprayed crystals are sensitive to the sun radiation and degraded in few days. In some Bt strains, the mature crystals are located between the exosporium and the spore coat. This special phenotype is called as spore-crystal association (SCA). In SCA phenotype strains, exosporium provides a natural defense, improving the crystal resistance to adverse environmental conditions. Artificial simulated SCA is an effective breeding strategy to develop new Bt capsule pesticide. Bt C15 is highly toxic to nematode and has a SCA phenotype. Here we presented the complete genome sequence of C15, which consists of a 5,637,049 bp circular chromosome, and 8 plasmids (from 240 314 bp to 3 188 bp). C15 genome encodes five novel insecticidal crystal potein (Cry) genes, including cry21-99, cry21-67, cry21-66, cry21-46 and cry-N. In this work, the cry21-99 gene was cloned and expressed in the acrystalliferous Btmutant BMB171. The purified Cry21-99 toxin showed high toxicity against Caenorhabditis elegans and Meloidogyne incognita. In addition, genes of other nematocidal factors and secondary metabolites were predicted. This work provides novel genetic resources for transgenic crops to control nematodes. Previously, we have identified the SCA controller in another B. thuringiensis SCA stain YBT-020 (serovar finitimus). The homolog genes of these SCA controller in C15 genome were predicted. However, the gene interrupted mutant maintained stable SCA phenotype. It indicates that the SCA formation in C15 is different from that of YBT-020, which represents a new SCA phenotype formation mechanism. This work provides clues for understanding the mechanism of SCA phenotype and development of novel high efficiency Bt capsule pesticides.