日本沼虾半胱氨酸蛋白酶抑制因子基因的克隆及其组织表达特征

Molecular cloning and initial function analysis of cystatin gene in Macrobrachium nipponense

半胱氨酸蛋白酶抑制因子(CST)是一种能够可逆结合并抑制半胱氨酸蛋白酶类的活性的蛋白,为了明确日本沼虾(Macrobrachium nipponense)CST基因(Mn CST)序列及其在日本沼虾卵巢中的作用,本研究利用RACE方法克隆获得日本沼虾Mn CST全长cDNA序列,利用qRT-PCR分析了其在不同组织和不同发育阶段的卵巢中的表达情况,并利用RNA干扰(RNAi)技术初步分析了Mn CST在日本沼虾卵巢发育中的作用。结果显示,Mn CST cDNA序列全长6199 bp,包含1183 bp 5′非编码区、2304 bp 3′非编码区和2709 bp开放阅读框,编码903个氨基酸残基,其氨基酸序列上有6个cystatin-like(半胱氨酸蛋白酶抑制因子样)结构域和42个磷酸化位点;Mn CST基因在各个组织中均有表达,肠中表达量最高;日本沼虾不同发育阶段的卵巢中均有Mn CST基因表达,Ⅳ期卵巢中表达量最高,Ⅱ期卵巢中表达量最低;Mn CST基因在日本沼虾卵巢中的表达变化与组织蛋白酶B(Cts B)和组织蛋白酶L(Cts L)基因的表达变化基本是一致的,但对卵黄蛋白原(Vg)基因的表达没有直接影响。Mn CST在日本沼虾卵巢中的作用可能主要是抑制Cts B和Cts L的活性,在日本沼虾卵巢发育过程中对Vg的水解起间接的调控作用。

Cystatins(CSTs)belong to a protein superfamily the members of which reversibly bind cysteine proteases and inhibit their activity.CSTs are divided into three families:family 1 CSTs are single domain cystatins that do not contain disulfide bridges and carbohydrate side chains;family 2 CSTs possess a single cystatin domain,but their structures have at least two intramolecular disulfide bridges;and family 3 CSTs display a higher degree of structural complexity characterized by the occurrence of multiple cystatin-like domains,each with two disulfide bridges at positions homologous to those in family 2 CSTs.CSTs widely occur in various vertebrates such as mammals,birds,and fishes.CSTs have also been identified in some invertebrates.In many cellular defense systems,the CSTs plays a role in preventing the excessive hydrolysis of cellular proteins by cysteine proteases,and regulate many metabolic processes that depend on cysteine proteases.However,the study of the role of CSTs in the gonads of animals has not yet been reported.Macrobrachium nipponense is a member of the Palaemonidae family of decapod crustaceans and is widely farmed in China because of its flavor and high nutritive and economic values.To breed superior variety of M.nipponense during actual production,it is necessary to study the characteristics and functions of the gonadal development-related genes of this species.The aims of this study were to identify the sequence information and function of CSTs in the ovary of M.nipponense.The full-length M.nipponense CST(MnCST)cDNA sequence was cloned using the rapid amplification of cDNA ends(RACE)technology;then,real-time quantitative PCR(RT-qPCR)was used to analyze the expression level of MnCST in different tissues and different developmental stages of the ovary;and finally,the role of MnCST in the ovary of M.nipponense was analyzed by RNA interference(RNAi)technology.The results showed that the full-length cDNA of MnCST was 6199 bp long,including 1183 bp at the 5′-UTR,2304 bp at the 3′-UTR,and a 2709 bp open reading frame encoding a peptide of 903 amino acids.The putative peptide contained six cystatin-like domains and 42 phosphorylation sites.Multiple sequence comparison of crustacean CST proteins indicated that MnCST has the highest similarity with the corresponding protein in Cherax quadricarinatus,while it has the lowest similarity with the corresponding protein in Eriocheir sinensis.Real-time quantitative PCR detected MnCST mRNA in nine different tissues of M.nipponense,and the maximum level was detected in the intestine.Moreover,MnCST mRNA was also detected in six developmental stages of the M.nipponense ovary;the maximum level was detected in the stage IV ovary,while the minimum level was detected in the stage II ovary.The results of RNAi showed that the expression change of the MnCST gene was basically consistent with the expression changes of cathepsin B(CtsB)and cathepsin L(CtsL)genes in the ovary of M.nipponense,while there was no effect on the expression of vitellogenin(Vg)gene.The role of MnCST in the ovary of M.nipponense is likely to inhibit the activities of cathepsins and indirectly regulate the hydrolysis of Vg during ovarian development.This study provides new insights into the role of CSTs in the gonads of crustaceans.