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【目的】构建优质陆地棉栽培品种冀228纤维均一化全长cDNA文库,降低纤维中存在的高峰度基因的拷贝数,提高发现纤维发育相关基因随机序列和稀有基因的效率,为公共数据库提供丰富的陆地棉EST资源。【方法】以优质陆地棉冀228开花后8—40 d纤维为材料,将纤维全长cDNA与Gateway供体载体p DONR222重组,构建了棉花纤维的非剪切型全长cDNA原始文库。利用均一化技术获得均一化全长cDNA文库,进而测序获得大量的EST序列。采用生物信息分析手段,对所测EST进行拼接、比对、COG功能注释和GO注释。【结果】构建了优质陆地棉冀228纤维均一化全长cDNA文库,该文库初级文库库容为1.06×107,初级文库的滴度为3.56×106 cfu·m L-1,平均片段长度为1.2 kb。q RT-PCR检测均一化程度结果表明,棉花2个高峰度表达基因在该文库中的表达量均下降了1 000倍。随机选取2 384个克隆进行单向测序,获得2 169条高质量的表达标签(EST),拼接成1 745个单一基因(Unigene);同源比对分析表明,70%的Unigenes与已知功能基因具有较高的同源性。基因进行COG功能分类结果显示,较多的COG功能分类集中在“翻译、核糖体结构和生物转化”、“碳水化合物运输与代谢”和“翻译后修饰、蛋白转移及蛋白伴侣”功能上。根据基因的GO注释结果,参与细胞构成、细胞器和膜构成的基因比例最高,参与细胞过程和新陈代谢等过程的基因比例较高,具有结合和催化功能的基因较多,这些基因可能在棉纤维发育中发挥比较重要的作用。【结论】构建了优质陆地棉栽培品种冀228纤维均一化全长cDNA文库,经文库质量检测、均一化程度检测和随机选取克隆的测序分析结果表明,文库的代表性和重组片段的完整性均达到了分离筛选目的基因的建库要求,整个文库有着很高的非冗余性。构建的cDNA文库具有既能获得与已知序列同源性很高的基因,又能挖掘出优质陆地棉冀228特有的基因或同源序列的作用,能够提高发现纤维发育相关基因随机序列和稀有基因的效率,将为公共数据库提供丰富的陆地棉EST资源。
【Objective】 The objective of this study was to construct a full-length cDNA library of high-quality upland cotton cultivars Ji 228 fiber, to reduce the copy number of high-peak genes present in the fiber, to increase the efficiency of discovering random sequences and rare genes of fiber-related genes and to enrich public databases Of upland cotton EST resources. [Method] The full-length cDNA of non-cleaved cotton full-length cDNA of cotton fiber was constructed based on the fiber of 8-40 d after flowering of high quality upland cotton Ji-228. The full-length cDNA was recombined with the Gateway donor vector pRON222. A uniform full-length cDNA library was obtained by using the homogenization technique, and a large number of EST sequences were obtained by sequencing. Bioinformatics analysis was used to make splicing, alignment, COG functional annotation and GO annotation on the ESTs tested. 【Result】 The results showed that the average library length was 1.2 kb and the average length of the primary library was 3.56 × 106 cfu · m L-1 . q RT-PCR detection of the degree of homogeneity results showed that the expression level of cotton two peaked genes in the library were reduced by 1 000 times. A total of 2 384 ESTs were randomly selected from 2 384 clones to obtain 1 169 high-quality ESTs, which were then ligated into 1 745 single genes (Unigene). Homologous alignment analysis showed that 70% of Unigenes hybridized with known functions Genes have high homology. The results of COG classification revealed that most of the COG functional categories were focused on the “translation, ribosome structure and bioconversion”, “carbohydrate transport and metabolism”, and posttranslational modifications, protein transfer and protein mate "Function. According to the gene GO annotation results, the genes involved in cell constitution, organelles and membranes constitute the highest proportion of genes involved in cellular processes and metabolic processes such as a higher proportion of genes with more binding and catalytic genes, these genes may be in the development of cotton fiber Play a more important role. 【Conclusion】 A full-length cDNA library of cultivars of upland cotton cultivars Ji 228 was constructed. The quality of the library, the degree of homogeneity and the randomly selected clones were analyzed by sequencing. The results showed that both the representativeness of the library and the integrity of the recombinant fragments The library has been isolated and screened for the purpose of gene screening requirements, the entire library has a high non-redundant. The constructed cDNA library can not only obtain the gene with high homology with the known sequence, but also dig out the gene or homologous sequence of the high-quality G. hirsutum 228, which can improve the discovery of the random sequence of the fiber development-related gene and rare The efficiency of the gene will provide a wealth of upland cotton EST resources for public databases.