金龟子绿僵菌中性海藻糖酶基因的克隆及其表达特性分析

Cloning and characterization of the neutral trehalase gene in Metarhizium anisopliae CQMa102

根据中性海藻糖酶NTL基因的同源序列设计引物,PCR扩增出杀蝗专一菌株———金龟子绿僵菌CQMa102NTL基因片段,利用5′_RACE和3′_RACE扩增出NTLcDNA的5′和3′端序列,经拼接得到CQMa102NTL基因cDNA全长。根据其全长cDNA序列,设计引物PCR扩增出CQMa102NTL的完整基因。为了解该基因的上游调控信息,采用PanhandlePolymeraseChainReactionAmplification方法扩增其上游序列。序列分析表明,CQMa102NTL全长DNA3484bp,cDNA全长2385bp,编码737个氨基酸的蛋白,推测蛋白分子量为83.1kD;含有3个内含子,包含一个依赖于cAMP的磷酸化作用位点(RRGS)和一个钙附着位点(DTDGNMQITIED);上游序列含有一个压力反应元件(CCCCT);与金龟子绿僵菌广谱性菌株ME1NTL的核苷酸序列和氨基酸序列分别具有93%和99%同源性,由此确定该序列为金龟子绿僵菌中性海藻糖酶基因序列。Southern杂交表明,NTL基因在CQMa102基因组中为单拷贝。Northern杂交表明,NTL基因转录出约2.5kb的mRNA单带,在液体培养条件下,对数生长前期表达水平最高,对数生长后期降到最低,进入稳定生长期后表达水平又有所提高。金龟子绿僵菌CQMa102中性海藻糖酶基因DNA全长和cDNA全长登录GenBank,登录号分别为:AY557613,AY557612。

The partial fragment of the neutral trehalase （NTL） in Metarhizium anisopliae CQMa102 was amplified by the polymerase chain reaction （PCR） with primers designed according to the sequences of the NTL in GenBank. The amplified fragment was cloned and sequenced. Based on the known sequence of NTL gene, the 5＇ and 3＇ rapid amplification of cDNA ends （RACE） were used to amplify the 5＇ and 3＇regions of the NTL cDNA, then the whole cDNA sequence of NTL gene in M. DNA was amplified by PCR. anisopliae CQMa102 was In order to obtain more obtained by combining the above sequences and its whole genomic regulatory information of the NTL, panhandle polymerase chain reaction strategy was used to amplify the 5＇ flanking sequences adjacent to the known sequence of the neutral trehalase gene. The sequence analysis shows that the DNA sequence is 3484bp in size, includes three introns, and the cDNA sequence is 2385bp in size, encodes a protein of 737 amino acid residues with a calculated Mr of 83.1kD, in which there is a cyclic adenosine 3＇, 5＇-monophosphate-dependent phosphorylation consensus site and a putative calcium binding site, which is consistent with a regulatory enzyme. Comparison of this sequence with the NTL of M. anisopliae ME1 in GenBank （GenBank Accession：AJ298019,AJ298020） shows that the nucleotide homology is as high as 93 %, and the amino acid homology comes up to 99 %. Southern analysis indicated that NTL gene was present as a single copy in this M. anisopliae strain. A single transcript of approximately 2.Skb was detected by Northern blot analysis .The NTL mRNA was present exponential throughout spore germination in rich medium, but accumulated to its highest level at the early stage of growth. Thereafter, the mRNA level declined at the late stage of exponential growth, but began to show an increase during the stationary phase of growth. A 982bp upstream sequence of NTL was amplified using panhandle PCR method, which contains one stress response element （STRE）. The NTL nucleotide sequence and its amino acid sequence have been accessed by GenBank （Accession： AY557613,AY557612）.