内蒙古螺旋藻HSP90基因的克隆及三亲接合表达载体构建

Cloning of a putative heat shock protein 90 gene from Spirulina innermongoliansis and construction of three pro-integration expression vector

内蒙古螺旋藻是一种原产于内蒙古碱湖,品质优良、中温适应型的螺旋藻品种,但因发现较晚,生长环境特殊等原因,对其分子遗传学等基础研究相对滞后。提高其耐热性能够扩大养殖范围,降低养殖成本,具有很大的应用价值。研究表明热激蛋白在藻类抗高温胁迫中起着重要作用。本研究采用PCR技术得到内蒙古螺旋藻HSP90基因序列,并对基因及其编码蛋白序列进行了初步分析;同时,根据蓝藻遗传转化需要,构建了三亲接合转移的穿梭表达载体,该载体起始密码子ATG与目的基因HSP90相距6 bp,含有进行三亲结合转移所需的蓝藻复制起始区、Bom基因、启动子(BetaP)、氯霉素抗性基因(Cm)及目的基因HSP90。研究结果为构建耐热内蒙古螺旋藻转基因新品系等奠定了基础。

Spirulina innermongoliansis is a high quality variety with normal temperature adaptability,which was found in salt lake in Inner Mongolia.Because of its later discovering history,and its special living conditions and other related reasons,S.innermongoliansis have not be widely studied and exploited in molecular genetics.Improving its heat resistance could expand its breeding range and reduce the farming costs.Some researches showed that heat shock protein played an important role in heat resistance of algae.This present study used PCR technology to gain HSP90 gene sequences in S.innermongoliansis,and then analyzed the gene sequence and its encoding protein gene sequence.Meanwhile,according to the genetic transformation of algae,we constructed a three pro-integration expression vector,whose initiation codon ATG was 6 bp away from HSP90 gene.Three pro-integration expression vector had a cyanobacterial replication origin,a Bom gene,a promoter（BetaP） and a chloramphenicol resistance gene（Cm）,which was essential to three pro-integration,then connected the gene HSP90.Therefore,this research was a necessary foundation to gain a new strain of S.innermongoliansis which had a higher heat resistance.