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针对罗汉果果实富含酚类、多糖、油脂和蛋白质等特点,从3种常用的RNA提取方法中优选出改良Trizol法,应用该法得到的RNA纯度高(OD260/280=2.01;OD260/230=2.02)、完整性好(RIN=9.50)、得率高(260μg·g-1)。以该法提取的RNA为模板,通过RACE和RT-PCR技术克隆罗汉果脱氢抗坏血酸还原酶基因全长,其长度为1 252 bp,开放阅读框(ORF)为819 bp,命名为SgDHAR,GenBank登录号为KC907731,编码一条272个氨基酸的肽链,理论分子量为30.217 7 kD,等电点为8.76。该蛋白与GenBank中已登录的DHAR序列比对显示与黄瓜同源性最高(87%)。应用实时荧光定量PCR对SgDHAR在罗汉果不同组织及不同倍性中的表达模式分析发现,该基因主要在果实和茎中表达,其次是花,根中的表达量最低;在三倍体中的表达量为二倍体的6.83倍,推测该基因可能参与了三倍体无籽罗汉果的败育过程。
According to the characteristics of Lo Han Guo fruits such as phenols, polysaccharides, oil and protein, the improved Trizol method was optimized from the three commonly used RNA extraction methods. The RNA obtained by this method has high purity (OD260 / 280 = 2.01; OD260 / 230 = 2.02), good integrity (RIN = 9.50), high yield (260μg · g-1). The RNA extracted from this method was cloned by RACE and RT-PCR, and its length was 1 252 bp and 819 bp with the open reading frame (ORF) of 819 bp, which were named as SgDHAR and GenBank. No. KC907731, encoding a 272 amino acid peptide chain, the theoretical molecular weight of 30.217 7 kD, isoelectric point of 8.76. The alignment of this protein with the DHAR sequence registered in GenBank showed the highest homology to cucumber (87%). Analysis of the expression pattern of SgDHAR in different tissues and different ploidies of Luohanguo by real-time fluorescence quantitative PCR showed that the gene was mainly expressed in fruits and stems, followed by flowers and roots, and the expression level in triploid The amount of 6.83 times the diploid, speculated that the gene may be involved in the seedless seedless triploid Luo Han Guo abortion.