罗汉果果实RNA的提取及SgDHAR基因的克隆与表达  

Extraction of total RNA and cloning of SgDHAR gene from Siraitia grosvenorii

作　　者：韦荣昌[1,2] 赵欢[2] 马小军[1,2] 密克[1] 莫长明[1] 潘丽梅[1] 白隆华[1] 唐其[1,3] 

机构地区：[1]广西药用植物园,广西南宁530023 [2]中国医学科学院、北京协和医学院药用植物研究所,北京100193 [3]湖南农业大学园艺园林学院,湖南长沙410128

出　　处：《药学学报》2014年第1期115-123,共9页Acta Pharmaceutica Sinica

基　　金：国家科技支撑计划课题资助项目(2011BAI01B03);国家自然科学基金资助项目(30960500);广西自然科学基金资助项目(2013GXNSFDA019021,2013GXNSFBA019170)

摘　　要：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。Total RNA was isolated from Siraitia grosvenorii fruit by the method of modified Trizol, according to S. grosvenorii fruit characteristics of rich phenols, polysaccharide, oil and proteins. The OD 26o/280, OD260/230, RNA integrity （RIN） and yield of the total RNA with this method were 2.01, 2.02, 9.50 and 260 ~tg-g-1, respectively. The open reading frame （ORF） of dehydroascorbate reductase （DHAR）, named as SgDHAR, was cloned by rapid amplification of cDNA ends （RACE） and RT-PCR method from S. grosvenorii. The GenBank accession number for this gene is KC907731. The SgDHAR gene contains a full-length cDNA of 1 252 bp including ORF of 819 bp and encodes a predicted protein of 272 amino acids. The molecular mass is 30.217 7 kD and the isoelectric point is 8.76. Homology comparison showed that it shared 87% nucleotide sequence homology with Cucumis sativus. Expression patterns using qRT-PCR analysis showed that SgDHAR was mainly expressed in fruit and stem, followed by flower, and was lowest in root, while the expression level was 6.83 times in triploid than that in diploid. Therefore, SgDHAR gene may be involved in abortion of triploid seedless S. grosvenorii.

关 键 词：罗汉果 改良Trizol法 DHAR基因 RACE 生物信息学 

分 类 号：R931[医药卫生—生药学]