两种色型黄粉虫抗冻蛋白cDNA克隆、序列分析与表达分析

Cloning,sequencing and expression analysis of antifreeze protein cDNAs from two color varieties of Tenebrio molitor(Coleoptera:Tenebrionidae)

产生抗冻蛋白(antifreeze proteins,AFPs)是大多数昆虫抵抗低温的一个重要策略。为给黄粉虫Tenebriomolitotr耐寒机理研究提供参考,本研究采用RT-PCR、5'RACE和3'RACE法克隆了黄、黑两种色型黄粉虫幼虫的抗冻蛋白基因Tm-afp,分析了其基因序列及所编码的氨基酸序列,并检测了其在这两种色型间的mRNA水平差异。结果表明:从黄、黑两种色型黄粉虫幼虫克隆出的Tm-afp cDNA全长分别为579bp和588bp,它们包含一个20bp的5'端非翻译区、一个402bp的开放阅读框和一个变异较大的3'端非翻译区,其碱基序列一致性为95%。由于这两个抗冻蛋白基因编码的蛋白成熟肽存在两个氨基酸差异:第35位(D→E)和130位(T→S),因此将其判定为黄粉虫抗冻蛋白基因Tm-afp的两个异构体,并分别命名为Tm-afp-1和Tm-afp-2。Tm-afp-1和Tm-afp-2编码的抗冻蛋白异构体(分别为Tm-afp-1和Tm-afp-2)信号肽之间有3个氨基酸差异:第2位(G→A)、9位(S→T)和27位(Y→N);其成熟肽的第一个氨基酸为谷氨酰胺,含8个高度保守的12aa短串联重复序列,每个重复序列的第2位和第8位为半胱氨酸。此外,Tm-afp-1和Tm-afp-2均属富含苏氨酸和半胱氨酸的昆虫抗冻蛋白,其二级结构均由大量的β-折叠和无规则卷曲组成,三级结构为8个特殊的右手β-螺旋,每一圈螺旋由12个氨基酸组成;在β-螺旋的一个侧面规则排列着由保守XCT形成的β折叠片层。同时,低温可以诱导黄、黑两种色型黄粉虫Tm-afp的表达,但长时间低温诱导下黑色型黄粉虫幼虫的Tm-afp表达量显著高于黄色型黄粉虫幼虫Tm-afp表达量。结果提示,黄粉虫种内不同色型间,抗冻蛋白基因Tm-afp可能存在多种异构体,并且黑色型黄粉虫Tm-afp比黄色型黄粉虫Tm-afp对低温的应答更强烈。这一研究结果为进一步探索黄粉虫的耐寒性机理提供了有益参考。

It is one of important strategies for many insects to produce antifreeze proteins（AFPs） to resist low temperature.In order to study the mechanism of cold resistance,an antifreeze protein gene Tm-afp was cloned from the larvae of yellow-and black-color varieties of Tenebrio molitor,the nucleotide sequence and the encoded amino acid sequence characteristics of Tm-afp were analyzed,and the mRNA levels were examined in the two varieties by reverse transcriptional PCR and rapid amplification of 5＇ and 3＇ cDNA ends.The results showed that the full cDNA sequences of Tm-afp cloned from the yellow-and black-color varieties of T.molitor were 579 bp and 588 bp in length,respectively.Both contain a 20 bp 5＇ non-coding region,a 402 bp open reading frame and a variant 3＇ non-coding region,and their nucleotide sequence identity is 95%.Because of two amino acid variation（D35→E35 and T130→S130） existing between the two mature peptides encoded by the two cDNA sequences,they were considered two isoforms of Tm-afp,i.e.,Tm-afp-1 and Tm-afp-2,respectively.Three amino acid variation（G2→A2,S9→T9 and Y27→N27） exists between signal peptides of the antifreeze protein isoforms（Tm-afp-1 and Tm-afp-2） encoded by Tm-afp-1 and Tm-afp-2.The 1st amino acid of the mature peptides of the two antifreeze protein isoforms is glutamine.Both the mature peptide sequences contain eight short-tandem repeats,each composed of twelve highly conservative amino acid residues.Furthermore,both the 2nd and 8th amino acids of each repeat are cysteines.In addition,Tm-afp-1 and Tm-afp-2 both belong to threonine-and cysteine-rich insect antifreeze proteins.Their secondary structure consists of beta sheets and random coils,and their tertiary structure has eight special right-handed beta helixes,composed of twelve amino acid residues per helix.Beta sheet,composed of conservative XCT,arrays on the lateral surface of the beta helixes.Meanwhile,low temperature up-regulated the expression of Tm-afp in yellow-and black-color varieties of T.molitor.But under long time of cold induction,the relative mRNA expression level of Tm-afp in the black-color variety of T.molitor was significantly higher than that in the yellow-color variety.The results suggest that Tm-afp among different color varieties of T.molitor may have several isoforms and Tm-afp in the black-color variety of T.molitor is more sensitive to cold than that in the yellow-color variety.This study may provide a useful reference to the further research on the molecular mechanisms of cold resistance in T.molitor.