Pt-CaM基因克隆及对菌核侧耳耐热性影响

Cloning of Pt-CaM Gene and Its Effect on Heat Tolerance of Pleurotus tuber-regium

通过保守序列比对、cDNA末端快速扩增技术和融合引物巢式PCR技术克隆菌核侧耳(Pleurotus tuber-regium)钙调蛋白(calmodulin,CaM)基因Pt-CaM。基因全长856bp,cDNA全长477bp,编码158个氨基酸。构建Pt-CaM的过表达和干涉载体,利用根癌农杆菌转化技术成功转化至菌核侧耳菌丝体中。在38℃高温培养下,过表达转化子的菌丝较野生型更致密,热激蛋白(heat shock protein)HSP70的表达量为野生型的3.89倍,超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)的活性为野生型的2.49倍和1.83倍;与野生型相比,干涉转化子菌丝生长速率降低,热胁迫损伤加重,其HSP70的表达量为野生型的0.77,SOD和CAT的活性无差别,过氧化物酶(peroxidase,POD)的活性为野生型的0.7。结果表明Pt-CaM可能通过影响HSP70的表达量和SOD、CAT和POD的活性参与菌核侧耳菌丝的耐热性调控。

The calmodulin(CaM) gene of Pleurotus tuber-regium Pt-CaM was cloned through conservative sequence alignment, rapid amplification of 3′cDNA ends( 3′RACE), and fusion primer and nested integrated PCR(FPNI-PCR). The full length of Pt-CaM was found to be 856 bp and its cDNA was 477 bp, encoding 158 amino acids. The overexpression and interference vectors of Pt-CaM were constructed and successfully transformed into P. tuber-regium mycelium by Agrobacterium tumefaciens mediated transformation, respectively.Compared with the wild-type strain, the Pt-CaM overexpression strain showed denser mycelia under 38 ℃, and its heat shock protein 70 gene HSP70 expression, superoxide dismutase(SOD) activity, and catalase(CAT) activity increased by 2. 89 times, 1. 49 times and 83 %, respectively. In contrast, the Pt-CaM interference strain exhibited a decrease in mycelial growth, aggravated heat stress damage, a decrease in HSP70 expression by 23 %, and a decrease in peroxidase(POD) activity by 30 % under 38 ℃. The activities of SOD and CAT remained the same in the interference strain as in the wild-type strain.The results suggested that Pt-CaM may participate in heat tolerance of P. tuber-regium mycelium via regulating HSP70 expression and activities of SOD, CAT and POD.