| Recently, it has been documented that the immune moleculars in invertebrates also possesss molecular polymorphism, which are similar to those of specific-immune molecules in vertebrate. And alternative splicing was supposed to be as an important mechanisms in forming molecular polymorphism. Hemocyanin is a multifunctional protein in the shrimp and other invertebrates, it has the the functions such as antiviral, antibacterial, phenoloxidase and other immunological activities. Interestingly, our previous finding indicated that shrimp L.vannamei hemocyanin showed single nucleotide polymorphisms(SNPs),.Furthermore, we occasionally found that alternative splicing (AS) might also occur in the transcription of mRNA. Therefore, an attempt was carried out to investigate the alternative splicing isoform of hemocyanin in Litopenaeus vannamei by using the methods include of RT-PCR, real-time PCR, semi-quantitative RT-PCR and bioinformatic,and the results of our studies are as follow:1 . Cloning of alternative splicing isoform cHE1The results of RT-PCR for 73kDa subunit of hemocyanin in heart tissues of shrimps revealed the existence of alternative splicing isoform cHE1(cDNA1 of hemocyanin), whose length of entire sequence was 2511bp, and the sequence was totally same with the DNA sequence of hemocyanin. It contains additional 296bp and 268bp sequences in N and C-terminal respcetively , comparing to the common isoform cHE(cDNA of hemocyanin) which includes 2000bp and encodes 663 amino acids. Then bioinformatics was further used, the results showed that the retained introns located outside of either the signal peptide or the copper-binding domains. And there is a stop code in the N-terminal of its intron, which led to the isoform cHE1 only encodes a presumed 26 amino acids peptide instead of the active hemocyanin protein.2 . mRNA expression anlysis of cHE1In order to further investigate the mRNA expression of cHE1, we designed RT-PCR experiments using the samples from shrimps of different developmental stages as well as various tissues of adult shrimps. The results revealed that cHE1 could express in different life-stages of shrimp. And its mRNA expression was observed in most of shrimp tissues including heart, muscle, gill, stomach, intestine and brain except hepatopancreas and lymph, while the common isoform seems to only express in hepatopancreas and lymph.3. The mRNA expression of cHE1 in bacteria or viroid-challenged shrimpsBased on the former findings, Vibrio Parahaemolyticus of Gram-negative bacteria, beta streptococcus of Gram-positive bacteria and viroid poly I:C were selected to inject the shrimps, then the mRNA expression of cHE1 was detected. The results showed an obvious up-regulation of cHE1 in 9-12h after infection, implying its potential immune function. For example, the shrimps respcetively produced the maximal quantity of cHE1 after the infection of Vibrio Parahaemolyticus, beta streptococcus and poly I:C at 12h, 9h, 12h, and these were 16 folds, 13 folds, 3.3 folds of the expressed cHE1 quantity in non-challenged shrimps, what's interesting, the results display a certain degree of inverse correlation between the expression of cHE1 mRNA and cHE mRNA. In conclution all these researches suggest the possible important immune function of cHE1 which is similar with the isoform of cHE, and the cHE1 may cooperate with cHE and play a role in shrimp immune defence. In summary, the present study showed that shrimp L.vannamei possess a novel hemocyanin alternative splicing isoform cHE1. Furthermore, it is possible involved in shrimp immune defence. These results will be contributed to investigate hemocyanin's diversity, understand its multifunctional mechanism and search a novel immune defence strategy against shrimp disease.
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