CRISPR-Cas9-mediated construction of a cotton CDPK mutant library for identification of insect-resistance genes  

作　　者：Fuqiu Wang Sijia Liang Guanying Wang Tianyu Hu Chunyang Fu Qiongqiong Wang Zhongping Xu Yibo Fan Lianlian Che Ling Min Bo Li Lu Long Wei Gao Xianlong Zhang Shuangxia Jin 

机构地区：[1]Hubei Hongshan Laboratory,National Key Laboratory of Crop Genetic Improvement,Huazhong Agricultural University,Wuhan 430070,China [2]Academy of Industry Innovation and Development,Huanghuai University,Zhumadian,Henan 463000,China [3]Xinjiang Key Laboratory of Crop Biotechnology,Institute of Nuclear and Biological Technology,Xinjiang Academy of Agricultural Sciences,Urumqi 830091 Xinjiang,China [4]National Key Laboratory of Cotton Bio-breeding and Integrated Utilization,School of Life Science,Henan University,Henan 475004,China [5]These authors contributed equally to this article

出　　处：《Plant Communications》2024年第11期23-40,共18页植物通讯（英文）

基　　金：Biological Breeding of Stress Tolerant and High Yield Cotton Varieties(2023ZD04040)to L.M.;National Natural Science Fund of China for Distinguished Young Scholars(32325039);National Natural Science Foundation of China(32272128)to S.J.,the National Natural Science Foundation of China(32401780);Key Scientific and Technological Project of Henan Province(222102110151)to S.L.;Major Science and Technology Project of Xinjiang Uygur Autonomous Region(2023A02003-2)to B.L.

摘　　要：Calcium-dependent protein kinases(CDPKs)act as key signal transduction enzymes in plants,especially in response to diverse stresses,including herbivory.In this study,a comprehensive analysis of the CDPK gene family in upland cotton revealed that GhCPKs are widely expressed in multiple cotton tissues and respond positively to various biotic and abiotic stresses.We developed a strategy for screening insect-resistance genes from a CRISPR-Cas9 mutant library of GhCPKs.The library was created using 246 single-guide RNAs targeting the GhCPK gene family to generate 518 independent T0 plants.The average target-gene coverage was 86.18%,the genome editing rate was 89.49%,and the editing heritability was 82%.An insect bioassay in the field led to identification of 14 GhCPK mutants that are resistant or susceptible to insects.The mutant that showed the clearest insect resistance,cpk33/74(in which the homologous genes GhCPK33 and GhCPK74 were knocked out),was selected for further study.Oral secretions from Spodoptera litura induced a rapid influx of Ca^(2+) in cpk33/74 leaves,resulting in a significant increase in jasmonic acid content.S-adenosylmethionine synthase is an important protein involved in plant stress response,and protein interaction experiments provided evidence for interactions of GhCPK33 and GhCPK74 with GhSAMS1 and GhSAM2.In addition,virus-induced gene silencing of GhSAMS1 and GhSAM2 in cotton impaired defense against S.litura.This study demonstrates an effective strategy for constructing a mutant library of a gene family in a polyploid plant species and offers valuable insights into the role of CDPKs in the interaction between plants and herbivorous insects.

关 键 词：COTTON CDPKS mutant library CRISPR-Cas9 Ca^(2+)influx insect resistance 

分 类 号：Q94[生物学—植物学]