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由EST获得全长cDNA对于结构基因组学和功能基因组学都是至关重要的,cDNA末端快速扩增技术RACE是该领域中的重要研究方法。利用BD SMART RACE技术扩增编码分泌天冬氨酸蛋白酶SA76基因的3’末端,将其与哈茨木霉cDNA文库中的SA76基因的EST序列进行序列拼接,获得2019bp的全长cDNA序列,其开放读码框长1593bp,5’非编码区266bp,3’非编码区201bp,编码530个氨基酸,有信号肽。哈茨木霉天冬氨酸蛋白酶基因与玉蜀黍赤霉、粗糙脉孢菌、球毛壳菌天冬氨酸蛋白酶基因的同源性分别为53%,37%,36%。利用BD SMART RACE技术首次从哈茨木霉中克隆天冬氨酸蛋白酶基因,为验证SA76基因的功能奠定基础,为进一步研究蛋白酶的作用机制及生物防治功能提供依据。图8参10。
Full-length cDNA obtained from EST is crucial for both structural genomics and functional genomics. The rapid amplification of cDNA ends (RACE) is an important research method in this field. The 3 ’end of the gene encoding aspartic protease SA76 was amplified using the BD SMART RACE technique and was spliced with the EST sequence of the SA76 gene in the T. harzianum cDNA library to obtain a 2019 bp full length cDNA sequence which was open The reading frame is 1593bp in length, 266bp in 5 ’non-coding region and 201bp in 3’ non-coding region. It encodes 530 amino acids and has signal peptide. The homology of T. harzianas aspartic proteinase gene to Gibberella zeae, Neurospora crassa, and Chaetomium globosum were 53%, 37% and 36%, respectively. It was the first time to clone aspartic protease gene from Trichoderma harzianum by using BD SMART RACE technology, which laid the foundation for the function verification of SA76 gene and provided the basis for further study of protease mechanism and biological control function. Figure 8 Reference 10.