Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (5): 813-824.doi: 10.3864/j.issn.0578-1752.2016.05.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

Establishment and Identification of a Normalized Full-Length cDNA Library of Upland Cotton Ji228

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   

  1. Cotton Research Institute, Hebei Academy of Agriculture and Forestry Sciences/Key Laboratory of Cotton Biology and Genetic Breeding in Huanghuaihai Semiarid Area, Ministry of Agriculture, Shijiazhuang 050051
  • Received:2015-07-31 Online:2016-03-01 Published:2016-03-01

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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

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