棉花纤维RNA提取方法的比较及酵母双杂交文库的构建

RNA Isolation and Yeast Two Hybrid Library Construction of Cotton Fibers

为了从棉花纤维中提取高质量的RNA用于构建酵母双杂交文库,采用4种不同的方法提取9天和19天棉花纤维的RNA。琼脂糖凝胶电泳和紫外分光光度法检测结果表明:热硼酸法适用于初生幼嫩纤维和次生加厚纤维的RNA提取,CTAB-PVP法仅适用于初生幼嫩纤维的RNA提取,异硫氰酸胍法和高盐高pH值法提取的RNA由于杂质含量较多而影响反转录效果。采用热硼酸法大量提取总RNA,纯化后的mRNA反转录为cDNA,通过重组将扩增的cDNA片段定向插入到pGADT7-RecAD克隆载体中,得到棉纤维9天和19天的酵母双杂交cDNA文库。2个文库的滴度分别是1.01×107cfu/mL和8×106cfu/mL,cDNA插入片段长度集中分布在250～2500bp之间。由此可以看出,构建的2个棉纤维文库质量较高,可用于棉花纤维素合成和调控途径的研究。

A high quality of RNA was essential for construction of yeast two hybrid library. In this work, four methods were performed to extract total RNA from samples of 9 dpa （clay post anthesis） and 19 dpa cotton fibers. RNA integrity and purity was detected by using agarose gel and spectrophotometer. As a result, the hot borate method worked well for 9 and 19 dpa cotton fibers, whereas CTAB-PVP was applicable only for 9 dpa fibers. As a comparison, both guanidinium thiocyanate and high ion-density methods were not suitable for the samples with high proteins and polysaccharides. The total RNA was extracted by hot borate extraction, the purified mRNA was reverse transcripted to eDNA. Then, the amplified cDNAs were inserted into pGADT7-Rec vector through highly efficient recombination system. Hence, the titers of the 9 and 19 dpa fibers eDNA library could respectively reach to 1.01×10^7 cfu/mL and 8×10^6 cfu/mL, and the eDNA insertions varied from 250 bp to 2500 bp. The two libraries with high quality could be used for the research of cellulose synthesis and regulation.