桑树花叶型萎缩病病株叶片组织中类似类病毒小分子RNA的检测与分析

为了能有效检测桑树中的类似类病毒小分子RNA(mscRNA),给解明mscRNA与桑树花叶型萎缩病发生的关系提供依据,以来自不同蚕区和不同季节发病桑园中的桑树花叶型萎缩病病株叶片为材料,采用LiCl法和DEAE纤维素柱层析法分别提取病叶组织中的mscRNA,然后利用往返聚丙烯酰胺凝胶电泳(Return-PAGE)进行检测鉴定,并对mscRNA在2mol/LLiCl溶液中的溶解性及该检测方法的灵敏度进行分析。结果显示:采用LiCl法和DEAE纤维素柱层析法都可以有效地提取阳性材料中含有的mscRNA;在溶解及不溶于2mol/LLiCl的阳性材料RNA样品中均含有丰富的mscRNA,mscRNA在LiCl溶液中的溶解性与已知类病毒的溶解性有很大的差异;当提取的病桑叶片组织总RNA量为480ng时,通过Return-PAGE可以检测到mscRNA,当总RNA量≤96ng时,已检测不到其中含有的mscRNA;在36株发病植株的叶片总RNA样品中,有7个样品检测出含有mscRNA,检出率约19%,在一些病症明显的植株样品中未检测出mscRNA,而在一些病症较轻的植株样品中却检测出mscRNA。检测结果提示:mscRNA与桑树花叶型萎缩病症的表现没有表现出明显的关联。

Determination and Analysis of Small Viroid-like RNA Isolated from Leaf Tissues of Mulberry with Mosaic Dwarf Disease

In order to determine mulberry small circular RNAs (mscRNAs) effectively so as to provide evidence for understanding the relationship between mscRNA and the occurrence of mulberry mosaic dwarf disease,the diseased leaves were collected from mulberry gardens with the mosaic dwarf disease in various sericultural areas and in different seasons for isolation of mscRNA using LiCl method and DEAE cellulose column chromatography method.The isolated mscRNAs were determined and identified by return polyacarylamide gel electrophoresis (Return-PAGE).Furthermore,the solubility of mscRNA in 2 mol/L LiCl and the sensitivity of Return-PAGE method were investigated.The results showed that both methods were effective in the extraction of mscRNAs from the positive samples.And both the soluble and insoluble fractions of the positive RNA samples contained abundant mscRNAs.The solubility of mscRNA in 2 mol/L LiCl was significantly different from the viroids reported previously.When the total RNA quantity extracted from diseased mulberry leaf tissues was 480 ng,mscRNA could be detected by Return-PAGE.When the total RNA was less than 96 ng,mscRNA could not be detected by Return-PAGE.mscRNAs were detected in total leaf RNAs from 7 of the 36 diseased mulberry trees,with a detection rate of about 19%.Nevertheless,no mscRNA was detected in leaf samples from the plants with obvious symptoms,while mscRNA was detected in leaf samples from plants with slight symptoms.These results suggest that mscRNA was not closely related to the symptoms of mulberry mosaic dwarf disease.