家蚕hsp24.3基因的克隆及功能研究

Cloning and Functional Study of Bombyx mori hsp24.3 Gene

【目的】对家蚕hsp24.3基因(Bmhsp24.3)的功能进行研究,为选育抗逆境家蚕品种提供基础材料和理论依据。【方法】在对家蚕基因组进行生物信息学分析的基础上,对家蚕低分子量热激蛋白基因Bmhsp24.3进行克隆,然后利用家蚕的芯片数据对Bmhsp24.3基因在5龄第3天幼虫不同组织中的表达模式进行分析,并利用半定量RT-PCR技术,分别对Bmhsp24.3基因在正常情况下和热刺激条件下家蚕5龄幼虫不同发育时期(1～6 d)后部丝腺中的表达状况以及10个不同家蚕品种5龄第3天幼虫丝腺中的表达情况进行了检测;最后将Bmhsp24.3基因进行原核表达,获得重组蛋白rBmHSP24.3,并进一步对该重组蛋白的功能进行了体外验证。【结果】Bmhsp24.3基因的编码区(CDS)长度为633 bp,编码210个氨基酸,为单外显子基因;RT-PCR检测发现该基因在家蚕的体壁、脂肪体以及丝腺组织中有较高水平表达,在其它组织中的表达不明显;同对照相比,不同家蚕品种以及不同发育时期的5龄幼虫在热刺激后,其后部丝腺中的Bmhsp24.3基因的表达显著升高。经Native-PAGE和SDS-PAGE分析表明,在高温条件下,表达获得的重组蛋白rBmHSP24.3能与硫氰酸酶形成稳定的复合物,使底物蛋白免受热刺激胁迫而变性,从而起着分子伴侣的作用。【结论】重组蛋白rBmHSP24.3在体外具有分子伴侣的功能,推测该蛋白在家蚕体内同样具有分子伴侣的功能,在家蚕的抗逆境适应过程中起重要作用。

【Objective】The function of Bombyx mori hsp24.3 gene was studied to provide a theoretical basis for raising Bombyx mori varieties with adverse resistance.【Method】Based on bioinformatic analysis of Bombyx mori’s genome database,the Bmhsp24.3 gene has been cloned,Using the silkworm’s microarray-based,the expression model of this gene in multiple silkworm tissues on day 3 of the fifth instar was analyzed.The expressional level analysis by using RT-PCR revealed that the Bmshsp24.3 expressed in Bombyx mori silk gland on day 3 of the fifth instar in different varieties or on days 1 to 6 of the fifth instar in P50 under normal raising condition and heat stimulating condition.The expression level of Bmhsp24.3 increased remarkably after heat stimulation.Finally,through recombinant expression,the recombinant protein rBmHSP24.3 was obtained and validated the function of this recominant protein.【Result】The CDS of the Bmhsp24.3 gene is 633 base pairs with no intron in it revealed by ESTs and coding 210 amino acids.This gene expressed in skin,fat body and silk gland of silkworm.The expression level in fat was remarkably different between male and female.Compared to the expression level of the Bmshsp24.3 under normal condition,the expression level increased remarkably after heat stimulation.The interactions of recombinant protein rBmHSP24.3 with Rhodanese under stress conditions were further analyzed.Both Native-PAGE and SDS-PAGE results demonstrated that the recombinant protein rBmHSP24.3 could form a stable complex with Rhodanese like a chaperone.Native-PAGE showed that the complex was detected,also free Rhodanese and recombinant protein rBmHSP24.3 could be observed.In order to verify the component of the complex,the complex was collected and applied to SDS-PAGE.Both proteins were delected in same lane,reasoning out the formatiom of the complex.Taken together,the interactions of recombinant protein rBmHSP24.3 with Rhodanese is a reversible interaction under heat stimulation.This experiment has proved that the recombinant protein rBmHSP24.3 was fully functional as a chaperone.It represented protection of proteins from irreversible aggregation.【Conclusion】The recombinant protein rBmHSP24.3 is fully active as a chaperone in vitro.It is concluded that the rBmHSP24.3 plays a role like a chaperone in vivo of Bombyx mori,and also shoulders an important role under adversity.