Abstract：
Calcium-dependent protein kinases (CDPKs) are pivotal signaling transduction enzymes in plants, especially responsive to diverse stress, including herbivory. In this study, through comprehensive analysis of CDPK gene family in upland cotton, we showed that GhCPKs are widely expressed in multiple tissues of cotton and positively respond to various biotic and abiotic stress. We developed a strategy for screening insect-resistant genes based on the CRISPR/Cas9 mutant library of GhCPKs. The library contains 82 members of the GhCPKs using 246 sgRNAs to generate 518 independent T0 plants. The coverage rate of target genes reached to 86.18%, the genome editing rate reached to 89.49%, and the editing heritability reached 82%. Through field insect bioassay, 14 GhCPK mutants resistant or susceptible to insect were identified. The most obvious insect-resistant mutant, cpk33/74 (simultaneously knocking out the homologous genes GhCPK33 and GhCPK74), was selected for further study. Oral secretions (OS) from Spodoptera litura induced a rapid influx of Ca2+ in cpk33/74 leaves, resulting in a significant increase in jasmonic acid (JA) content. S-adenosylmethionine synthase (SAMS) is an important protein involved in plant stress response, protein interaction experiments provided evidence of interactions between GhCPK33 and GhCPK74 with GhSAMS1 and GhSAM2, respectively. Additionally, silencing GhSAMS1 and GhSAM2 in cotton using VIGS resulted in decreased defense against S. litura. This study provides an effective strategy for constructing a mutant library of gene families in polyploid plant species and valuable insights into the role of CDPKs in the interaction between plants and herbivorous insects.
摘要：
钙依赖性蛋白激酶(CDPKs)是植物中关键的信号转导酶,特别是对各种压力的反应，包括食草动物.在这项研究中,通过对陆地棉CDPK基因家族的综合分析,我们表明GhCPKs在棉花的多个组织中广泛表达，并对各种生物和非生物胁迫产生积极响应。我们开发了一种基于GhCPK的CRISPR/Cas9突变文库筛选昆虫抗性基因的策略。该文库含有82个GhCPK成员,使用246个sgRNA产生518个独立的T0植物。目标基因覆盖率达到86.18%,基因组编辑率达到89.49%,编辑遗传力达到82%。通过野外昆虫生物测定，鉴定了14个对昆虫具有抗性或易感的GhCPK突变体。最明显的抗虫突变体,cpk33/74（同时敲除同源基因GhCPK33和GhCPK74），被选作进一步研究。斜纹夜蛾的口腔分泌物（OS）诱导cpk33/74叶片中Ca2的快速流入，导致茉莉酸（JA）含量显着增加。S-腺苷甲硫氨酸合成酶(SAMS)是参与植物逆境胁迫反应的重要蛋白,蛋白质相互作用实验提供了GhCPK33和GhCPK74分别与GhSAMS1和GhSAM2相互作用的证据。此外,使用VIGS沉默棉花中的GhSAMS1和GhSAM2会导致对斜纹链球菌的防御能力下降。本研究为构建多倍体植物基因家族突变文库提供了有效的策略，并为CDPKs在植物与食草昆虫相互作用中的作用提供了有价值的见解。
