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

摘要/Abstract

摘要： 【目的】深入了解花生胚发育的分子机制，获得花生胚发育相关基因。【方法】以优质花生品种闽花6号为材料，分别取花生果针入土后10、20、30、40、50和60 d（day after pegging，DAP）的胚作为试验材料，用CTAB法提取花生胚总RNA，采用SMART（Switching Mechanism At 5’ end of RNA Transcript）技术合成双链cDNA，经SfiⅠ酶切后胶回收纯化双链cDNA，连接到质粒载体pDNR-LIB上，电击转化大肠杆菌DH5α感受态细胞，构建不同发育时期花生胚混合全长cDNA文库，进行小规模测序。采用生物信息学分析手段对所获得的EST进行功能注释。【结果】构建了不同发育时期花生胚混合全长cDNA文库，文库的库容为3.5×106 cfu/mL，重组率95.8%，插入片段长度在500—2 000 bp，平均长度超过1 000 bp。随机挑取 60 个克隆进行5′端测序，共得到60条高质量的EST序列，BLASTX结果显示，有39条序列（65%）与GenBank中大豆、花生、苜蓿等植物上已公布的序列有较高同源性，其中，有32条具有已知或推测的功能，7条序列功能未知。其余21条（35%）序列在NCBI的非冗余蛋白数据库中没有找到与之相匹配的序列，可能为花生新的EST序列。利用BLAST2GO对所得序列进行GO注释分析，结果表明，参与抗逆与防御、蛋白质合成与运输、油脂合成与代谢、转录与调控以及种子萌发、休眠、胚发育相关的基因比较丰富，还有部分基因参与信号传导以及光形态建成等过程。KEGG代谢途径分析表明，随机测序所获得的序列中主要包括α-亚麻酸代谢和亚油酸代谢。【结论】利用SMART技术成功构建了不同发育时期花生胚混合全长cDNA文库；小规模EST测序分析获得了部分与花生胚发育相关的基因，如亚油酸9S-脂肪氧合酶、油体蛋白、锌指蛋白、热休克蛋白90、胚胎发育晚期丰富蛋白、脂肪氧合酶以及DREB转录因子等。

关键词: 花生 , 胚 , cDNA文库 , SMART技术 , 表达序列标签

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

陈华1, 2, 邓烨1, 2, 张冲1, 蔡铁城1, 2, 郑奕雄1, 2, 3, 庄伟建1, 2. 不同发育时期花生胚混合全长cDNA文库的构建与分析[J]. 中国农业科学, 2014, 47(11): 2272-2280.

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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