由于新疆野苹果(Malus sieversii)果实富含多糖、多酚、可溶性果胶及其他次生代谢物质,导致果实的总RNA提取相对比较困难,质量较差。因此,本试验以不同发育时期的新疆野苹果的果实为材料,采用TaKaRa试剂盒、全氏金试剂盒、田伟等改良的CTAB法、本试验改良的CTAB法(一)和CTAB法(二)这5种方法来摸索提取野苹果果实的总RNA的有效方法。结果表明:2种试剂盒均无法从野苹果果实中提取出RNA;通过3种改良CTAB法提取的RNA,在琼脂糖凝胶上得到清晰的28S和18S条带,并且纯度均较高,OD260/OD280值在1.8~2.2之间、OD260/OD230值大于2.0。但是只有本试验改良CTAB法(一)获得较高的RNA产量,从5个不同发育时期的野苹果果实中提取的RNA平均值在116.79~474.76 μg·g-1。改良CTAB法(一)通过延长LiCl沉淀时间,将-20 ℃放置15 min改为4 ℃放置过夜(时间不超过16 h),可以有效地除去RNA中的杂质污染,使得RNA充分沉淀。这种改良方法耗时相对较长,需要2 d,但是方法易于操作且提取成本较低。因此,改良CTAB法(一)是适用于提取野苹果果实(尤其是发育晚期)总RNA的有效方法,完全可以满足后期的分子生物学试验所需。
Malus sieversii fruits are rich in polysaccharides,polyphenols,soluble pectin and other secondary metabolites. Therefore,it is relatively difficult to extract the total RNA from M.sieversii fruits. In order to choose an effective extraction method,five RNA extraction methods including the TaKaRa Kit,Trans Kit,improved CTAB method by Tian Wei,modified CTAB method I and CTAB method Ⅱwere compared using the fruits at five different developmental stages. Results showed that neither kits method was able to be used to extract the RNA from M.sieversii fruits. The quality of total RNA extracted with three modified CTAB methods was good,with two distinct electrophoresis bands of 28S and 18S rRNA,the OD260/OD280 values ranged from 1.8 to 2.2,and the OD260/OD230 value was higher than 2.0. But only the modified CTAB method I was an effective method to yield high quantity of total RNA. The average extraction values of fruits at five different developmental stages were ranged from 116.79 μg·g-1 to 474.76 μg·g-1. For the modified CTAB method I,the impurity in the RNA was effectively removed by extending LiCl precipitation time and placing extraction under 4 ℃ for overnight aging (not longer than 16 hours)instead of -20 ℃ for 15 min. Although this method took 2 days,the method was easy to operate,and the extraction cost was low. Therefore,the modified CTAB method I is an effective method for total RNA extraction from M.sieversii fruits (especially for the ripe fruits),which could be used in further molecular biology research.
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