不同发育时期花生胚混合全长cDNA文库的构建与分析

doi:10.3864/j.issn.0578-1752.2014.11.021

Abstract

Abstract: 【Objective】 The objective of this study is to understand the molecular mechanism of peanut embryo development and obtain important genes related to peanut embryo development. 【Method】 Using peanut variety Minhua 6 as the experimental material, embryos on 10, 20, 30, 40, 50, and 60th day after pegging were sampled. Total RNA was extracted by improved CTAB method. Double strand cDNA was synthesized based on SMART technique. The purified dscDNA was ligated to pDNR-LIB vector digested by SfiⅠ and transformed into DH5α by electroporation to construct a full-length cDNA library of peanut embryos at different developmental stages. Bioinformatics analysis was performed following small-scale EST sequencing.【Result】A successful full-length cDNA library of peanut embryos at different development stages was constructed. The titer of unamplified cDNA library was about 3.5×106cfu/mL. The average cDNA inserts were more than 1 000 bp with a recombination frequency of 95.8%. Small-scale plasmid extraction and subsequent sequencing resulted in 60 ESTs, which were used for further analysis. BLASTX analysis showed that 39 sequences (65% of total sequences) had high similarity with reported genes in Glycine max, Arachis hypogaea, Medicago truncatula, etc. on NCBI with 32 sequences having known or putative functions and functions of other 7 sequences were unclear. The other 21 (35% of total sequences) could not find similarity with known genes in NCBI, which may be novel genes for peanut. GO annotation was performed with BLAST2GO software and the results revealed that the ESTs generated in this study mainly included responsive to stresses and defenses, protein synthesis and transport, lipid synthesis and metabolism, transcription and regulation, seed germination, dormancy and embryo development related genes. Besides, some genes were involved in signal transduction and light morphogenesis process. KEGG pathway analysis showed that the ESTs generated by randomly sequencing in this study mainly involved in alpha-linolenic acid metabolism and linoleic acid metabolism.【Conclusion】A high-quality full-length cDNA library of peanut embryos was constructed successfully using SMART method and some genes related to peanut embryo development were got, such as linoleate 9S-lipoxygenase-4-like, oleosin, zinc finger protein, heat shock protein 90, late embryogenesis abundant protein group 2, lipoxygenase-10, and putative DREB transcription factor, etc.

Key words: Arachis hypogaea L. , embryo , cDNA library , SMART , EST

CHEN Hua-1, 2 , DENG Ye-1, 2 , ZHANG Chong-1, CAI Tie-Cheng-1, 2 , ZHENG Yi-Xiong-1, 2 , 3 , ZHUANG Wei-Jian-1, 2 . Construction and Analysis of a Full-Length cDNA Library of Peanut Embryos at Different Developmental Stages[J].Scientia Agricultura Sinica, 2014, 47(11): 2272-2280.

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