应用“单抗组学”技术筛选棉纤维发育关键基因和转录因子

The applicant’s laboratory has developed the world's first monoclonal antibody library targeting whole protein profile of cotton in the early cooperation with an enterprise of monoclonal antibodies production and created successfully cotton protein antibody microarrays which contain 46,000 monoclonal antibodies, and has obtained 372 antibody signal points through the comparison of cotton fiber mutant.(fl) and wild-type cotton. The project intends to (1) perform screening for various cotton lintless and fuzzless fiber mutant and their wild-type by using the antibody microarrays. The corresponding protein targets of those antibody signals will be identified by immunoprecipitation and mass spectrometry. (2) Obtain protein antibodies against the key genes and transcription factors related to.cotton fiber development through screening the antibody microarrays with polypeptides or proteins expressed in vitro. (3) Select candidate genes and transcription factors for the further gene editing and genetic transformation of cotton based on above results. And then the corresponding antibodies will be used to perform Co-Immunoprecipitation/mass spectrum, chromatin immunoprecipitation.assay and High-seq sequencing, and immunohistochemistry (IHC) to get information of key genes on interacting proteins, regulating gene targets, and tissue expression and location. The completion of this project will further clarify the molecular mechanism on the regulation of key genes on cotton fiber development and epidermal cell differentiation. It will also set up a brand-new application and detection system for the study of gene regulation during plant development.

申请人所在实验室前期与单克隆抗体生产企业合作开发出第一个针对棉花全蛋白组的单克隆抗体文库，制作成包含46,000个单克隆抗体的棉花蛋白抗体芯片，利用该芯片比较棉花无纤维突变体fl和野生型获得超过372个显著性差异信号点。本项目拟在此基础上利用该抗体芯片（1）比较其他长、短绒等不同纤维发育突变体与野生型棉花的抗体差异信号，通过免疫沉淀和质谱鉴定差异蛋白质；（2）通过化学合成多肽或体外表达纤维发育相关基因或转录因子蛋白筛选抗体库芯片获得特异性抗体；（3）综合前两项结果选择重要基因或转录因子进行棉花基因编辑和遗传转化，并利用对应抗体进行蛋白质免疫共沉淀串联质谱、染色质免疫共沉淀高通量测序和免疫组化分析，解析关键基因或转录因子的蛋白相互作用、调控靶标和组织表达与定位等信息。本研究将会进一步阐明关键基因调控棉纤维发育的分子机制，也将为植物组织器官发育的基因表达调控研究建立一套新的应用和检测系统。

该研究项目基于实验室前期与单克隆抗体生产企业合作开发出第一个针对棉花全蛋白组的单克隆抗体文库，制作成包含46,000个单克隆抗体的棉花蛋白抗体芯片，经过ELISA测定抽检188个克隆的细胞上清产生的抗体浓度，97%的克隆产生的抗体浓度>5ug/ml；挑取100克隆进行CDR克隆测序及3000个克隆的混合扩增测序，得到抗体的CDR多样性为90.4%; 通过240个抗体的WB实验鉴定其识别独立蛋白至少为66，推算其覆盖率为27.5%, 即推算最终抗体库识别的独立蛋白数为33840*27.5%=9306个；通过不同发育阶段的胚珠的芯片筛选，并结合其转录组数据进行 WGCNA 的分析，挑选出 1200 个抗体与该发育过程相关，并对其进行了 217 个蛋白的鉴定，同时进行了部分蛋白在不同发育阶段的胚珠中的表达分析, 分析并鉴定到几个与棉纤维起始和延长高度相关的潜在功能蛋白。本研究为研究棉花和纤维发育建立一套新的应用和检测系统，具有重要的潜在应用价值。