冀228纤维均一化全长cDNA文库的构建与鉴定分析

doi:10.3864/j.issn.0578-1752.2016.05.001

摘要/Abstract

摘要： 【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pDONR222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·mL^-1，平均片段长度为1.2 kb。qRT-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。

关键词: 陆地棉, 纤维, 均一化, cDNA文库

Abstract: 【Objective】 To improve the efficiency of finding random sequence and rare genes that related to fiber development and provide abundant ESTs of upland cotton, we constructed a normalized fiber cDNA library of elite Gossypium hirsutum cv. Ji228 by decreasing copy numbers of the genes of high degree. 【Method】 To construct the original full-length cDNA library, full-length cDNA of 8 to 40 DPA (days post anther) fibers of elite upland cotton Ji228 were recombined with Gateway donor vector pDONR222. A normalized full-length cDNA library of fiber was established by normalization method, and a large number of ESTs were obtained by sequencing. For bioinformatics analysis, alignment, assembling, COG functional annotation and GO annotation were done. 【Result】 A normalized full-length cDNA library of fiber from elite upland cotton Ji228 was established. The capacity was 1.06×10⁷. The titer of primary library was 3.56×10⁶ cfu·mL^-1, and the average insertion size was 1.2 kb. Results of qRT-PCR demonstrated that normalization produced about 1 000 fold average reduction of two high abundant cotton genes. Random selected 2 384 clones were sequenced. 2 169 high-quality ESTs were obtained and assembled into 1 745 unigenes. Homologous alignments showed that approximately 70% of the unigenes had high homology with known genes. COG function classification revealed that COG function annotation mainly involved ‘translation, ribosomal structure and biogenesis’, ‘carbohydrate transport and metabolism’ and ‘posttranslational modification, protein turnover, chaperones’. According to the results of gene GO annotation, the genes that were involved in cellularity, constitution of organelle and cytomembrane had the highest proportion, moreover, the ratios were higher in cellular and metabolic processes, and genes that function in combining and catalysis also had higher proportions. These genes may play an important role in cotton fiber development. 【Conclusion】 In this study, we established a normalized full-length cDNA library of fiber from elite G. hirsutum cv. Ji228. Results of the library quality testing, the degree of homogenization testing and random cloning sequencing showed that the representation of the library and the integrity of the recombinant fragments met the requirements of library construction. The library had high redundancy. The results of one-by-one comparison of some unigenes demonstrated that the clones obtained from our cDNA library constructed in this study contained genes which not only had high homology with known sequences but also with unique or other sequences in Ji228. These results improved the efficiency of finding random sequence and rare genes that related to fiber development and provided abundant ESTs of upland cotton.

Key words: upland cotton, fiber, normalization, cDNA library

迟吉娜，蔡肖，张建宏，甄军波，刘琳琳，田海燕，唐丽媛，刘存敬，崔瑞敏，张香云. 冀228纤维均一化全长cDNA文库的构建与鉴定分析[J]. 中国农业科学, 2016, 49(5): 813-824.

CHI Ji-na, CAI Xiao, ZHANG Jian-hong, ZHEN Jun-bo, LIU Lin-lin, TIAN Hai-yan, TANG Li-yuan, LIU Cun-jing, CUI Rui-min, ZHANG Xiang-yun. Establishment and Identification of a Normalized Full-Length cDNA Library of Upland Cotton Ji228[J]. Scientia Agricultura Sinica, 2016, 49(5): 813-824.

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