球毛壳菌甘油醛-3-磷酸脱氢酶基因克隆及特性分析

Cloning and characterization of glyceraldehyde-3-phosphate dehydrogenase gene from Chaetomium globosum

用粗糙脉孢菌(Neurospora crassa,XP_327967)和菜豆炭疽病菌(Colletotrichum lindemuthianu,P35143)的甘油醛_3_磷酸脱氢酶基因(Glyceraldehyde 3_phosphatedehydrogenase,GAPDH)氨基酸序列对球毛壳菌(Chaetomium globosum)菌丝ESTs序列本地数据库进行tBlastn检索,获得了球毛壳菌GAPDH全长cDNA序列。该序列长1240bp,开放阅读框1014bp,编码337个氨基酸组成的多肽,蛋白分子量为36.1kD。用PCR方法克隆了该基因的DNA序列,序列长为1556bp,由2个内含子和3个外显子组成。BlastP同源性分析表明该基因与鹅掌柄孢壳(Podosporaanserine)同源性最高为95%;与米曲霉(Aspergillusoryzae)同源性最低为87%。GAPDH酵母转化子生物功能分析表明转化子对Na2CO3和高温有高的耐受性,证明GAPDH为抗胁迫基因。该基因的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY522719,AY593253,AAS01412)。

The amino acid sequence of Glyceraldehyde 3-phosphate dehydrogenase （GAPDH） gene from Neurospora crassa （XP-327967） and Colletotrichum lindemuthianu （P35143） were subjected to local tBlastn searching against the ESTs local datebase of Chaetomium globosum. The full length cDNA sequence of 1240bp encoding GAPDH gene with an open reading frame of 1014bp and encoding 337 amino acids was obtained. The protein molecular weight was 36. lkD. The DNA sequence of GAPDH gene was obtained through PCR amplification with specific primers of cDNA 5＇ and 3＇ untranslated region. The analysis of DNA sequence indicated that GAPDH gene have 3 exons and 2 introns. The BlastP analysis revealed that amino acids sequence of GAPDH gene from C. globosum shared 95 % high similarity with Podospora anserin and 87 % low similarty with Aspergillus oryzae. A transgenic yeast harboring C. globosum GAPDH was generated under the control of a constitutively expressed GAL promoter. The results from biofunctional analyses of GAPDH yeast transformants showed that GAPDH yeast transformants had significantly higher resistance to Na2CO3 and heat stresses. The cDNA, DNA and deduced amino acid sequence of GAPDH gene were accepted by GenBank （accession numbers ： AY522719, AY593253, AAS01412）.