论文部分内容阅读
凝集素在食用菌的生长发育、抗病虫等逆境及抗肿瘤等方面发挥着重要的作用,但是关于糙皮侧耳凝集素基因的抗逆功能研究还比较薄弱。因此,本文利用PCR技术克隆得到糙皮侧耳子实体凝集素polectin2基因及其启动子序列,测序和生物信息学结果显示该基因不含内含子,完整开放阅读框为408bp,编码136个氨基酸,预测蛋白分子量15.3k Da;启动子polectinpro序列长度为927bp,生物数据库PLACE分析结果表明polectinpro含有低温、干旱胁迫诱导和病程相关等抗逆元件。然后,构建含有GFP融合蛋白的重组植物表达载体pCAMBIA1302-polectinpro-gfp,并进行烟草遗传转化,结果证明该启动子能够在低温诱导下驱动gfp基因表达。同时成功构建了含有polectin2的重组植物表达载体pCAMBIA1301-polectin2并进行了烟草遗传转化;对阳性转化植株T1代和野生型种子进行低温胁迫实验,结果表明转基因种子萌发率明显高于野生型。综上所述,推测polectin2基因具有耐低温功能,且其启动子具有低温诱导活性。上述结果为进一步利用子实体凝集素polectin2基因提高食用菌的耐低温等抗逆功能提供了理论依据和技术支持。
Lectins play an important role in the growth and development of edible fungi, adversities such as disease and insect resistance and anti-tumor, but the research on the anti-retrogradation function of Pleurotus ostreatus genes is still relatively weak. Therefore, we cloned the lectin polectin2 gene and its promoter sequence by PCR. The sequencing and bioinformatics results showed that the gene contained no introns. The complete open reading frame was 408 bp, encoding a protein of 136 amino acids, The molecular weight of the predicted protein was 15.3 kDa. The length of the promoter polectinpro was 927 bp. The bioinformatics analysis of PLACE showed that polectinpro contained low temperature, drought-stress-induced and disease-resistant components. Then, a recombinant plant expression vector pCAMBIA1302-polectinpro-gfp containing GFP fusion protein was constructed and transformed into tobacco. The results showed that the promoter can drive gfp gene expression under the induction of low temperature. At the same time, a recombinant plant expression vector pCAMBIA1301-polectin2 containing polectin2 was successfully constructed and transformed into tobacco. The transgenic plants T1 and wild-type seeds were subjected to low temperature stress. The results showed that the germination rate of transgenic seeds was significantly higher than that of the wild type. In summary, we speculated that polectin2 gene has low temperature resistance, and its promoter has low temperature-induced activity. These results provide theoretical basis and technical support for further utilization of the fruiting body lectin polectin2 gene to improve the low temperature resistance of edible fungi.