千年桐SAD基因克隆与分析及其丝状真菌表达载体构建

以发育中的千年桐种子总RNA为模板,通过RT-PCR方法扩增得到硬脂酰脱饱和酶基因SAD的cDNA序列。该序列长度为1 191 bp,编码396个氨基酸。推测的分子量为45 541.01 u,等电点pI为6.05。BLAST分析表明,该cDNA序列与其它已登录的SAD基因cDNA序列一致性最高可达93.1%;编码的氨基酸蛋白序列性一致最高为89%。同时,构建了由构巢曲霉3-磷酸甘油醛脱氢酶基因的gpdA启动子驱动的丝状真菌表达载体,通过冻融法转入农杆菌中,PCR鉴定表明,pBAR-SAD已转入农杆菌EHA105中,成功构建了农杆菌工程菌株。

Clonging and Characterization of SAD from Vernicia montana and Construction of Filamentous Fungi Expression Vector

The total RNA was extracted from the developing seeds of Vernicia montana,and the sequence of SAD was obtained by RT-PCR reaction,which was 1 191 bp long and encoded a protein of 396 amino acides with Mr=45 541.01 u and pI=6.05.The deduced nucleotides had 93.1% identity in comparison with the reported datas and 89% identity with other amino acids.A filamentous fungi expression vector,which was controlled by promoter of Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene,was constructed.The recombinant plasmid DNA was transformed into Agrobacterium tumefaciens by the Freeze-Thaw Method.It determined that pBAR-SAD has been transformed into EHA105 by PCR.