甜菜M14品系FIE基因的鉴定及花期表达cDNA文库的构建

【作者】 郑天然；

【导师】 王桂芝；

【作者基本信息】 黑龙江大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 FIE基因首先在拟南芥中被分离，它控制着非受精种子的发育，可能是无融合生殖的组件之一。根据FIE的保守性，本实验运用保守序列法，尝试在甜菜M14品系中分离同源基因。根据FIE的基因序列设计PCR引物，以拟南芥总DNA为模板进行PCR扩增FIE特异性片段，并连接到pGEM-T Easy载体上转化E.coli JM109菌种。酶切鉴定及测序分析结果表明该克隆为拟南芥FIE基因特异片段，与拟南芥FIE基因比较同源性达99.8％，可以作为探针使用。以FIE特异片段为模板，用地高锌标记探针进行Southern杂交，结果表明甜菜M14品系中含有FIE的同源基因片段。以甜菜M14花蕾为材料，提取总RNA，分离mRNA，合成双链cDNA，通过RT-PCR法克隆得到了甜菜M14品系FIE基因406bp大小的特异片段。为了得到全长基因，采用cDNA文库快速构建法制备了M14花蕾cDNA文库。以后通过cDNA文库可直接筛选甜菜M14品系FIE等目的基因。更多还原

【Abstract】 FIE gene, which was first isolated in Arabidopsis thaliana, controls the development of fertilization-independent endosperm, and it might be one of components in apomixis. According to the conservation of FIE, We use methods of conservative sequence and attempt to find homological genes in Beta vulgaris M14.PCR primers were designed according to FIE gene sequence,than amplified FIE specific fragment of total DNA in Arabidopsis thaliana, linked to pGEM-T Easy Vector and transformed E.coli JM109. Restriction analysis and sequence analysis have proved that the colne product is FIE specific fragment of Arabidopsis thaliana. Comparing with FIE gene of Arabidopsis thaliana shared 99.8% homology at nucleotide level. It can be used as probes.By using FIE specific fragment as template,probes were labeled by digoxigenin(DIG). The results of Southern hybridization with genomic DNA in Beta vulgaris M14 suggest that M14 has homological genes of FIE. Total RNA was extracted from the bud of Beta vulgaris M14 followed by purification of mRNA, synthesis of the first strand and the second strand cDNA and obtained FIE specific fragment of 406bp of Beta vulgaris M14 by using RT-PCR. In order to obtain the gene of full length, the cDNA Library of M14 bud was constructed through highly efficient and simplified method. The resultant cDNA library will be used for the screeing of target genes of Beta vulgaris M14.更多还原