适于cDNA-AFLP的黑木相思组培苗总RNA快速提取方法的建立

针对黑木相思富含多酚、多糖等次生代谢物质的特点,以其组培苗为试材,采用改良CTAB裂解法、硼砂-SDS裂解法、改良异硫氰酸胍裂解法、CTAB-异硫氰酸胍裂解法、改良CTAB-异硫氰酸胍裂解法等5种不同的提取方法提取其总RNA.结果发现:改良CTAB-异硫氰酸胍裂解法能有效地控制多酚、多糖、蛋白质以及DNA等对所提取总RNA的污染,所得总RNA质量高、完整性好.紫外分光光度检测,A260/A280比值为1.96～2.00,A260/A230比值1.98～2.20,总RNA得率为118.4～213.6μg.g-1,电泳检测,28SrRNA亮度约为18SrRNA的2倍,所提RNA质量可以满足dscDNA合成和cDNA-AFLP等后续分子操作要求.

A Fast Method Suited to Extract Total RNA Used to cDNA-AFLP from Plantlets of Acacia melanoxylon Cultured in Vitro

The borax-SDS method, modified CTAB method, modified guanidinium isothiocyanate method,CTAB-guanidinium isothiocyanate method, modified CTAB-guanidinium isothiocyanate method were used to extract the total RNA from the plantlets of Acacia melanoxylon cultured in vitro, in which contain a lot of polysaccharide, polyphenol and secondary metabolites. The modified CTAB-guanidinium isothiocyanate method can efficiently remove polyphenol, polysaccharide, proteins and DNA that often contaminate with RNA. The high-quality and integrity total RNA acquired the 28S rRNA, which is approximately two times as light as the 18S rRNA,with 1.96～2.00 of A_(260)/A_(280) ratio and 1.98～2.20 of A_(260)/A_(230) ratio, and the total RNA yield of 118.4～213.6 μg·g~(-1), and the quality of extracted RNA meet the requirement of subsequent molecular biology reactions as in dscDNA synthesis and cDNA-AFLP.