Study On The Function Of MnMats, MnTaiman And A Hypervariable C-type Lectin In The Innate Immunity Of Macrobrachium Nipponense

Macrobrachium nipponense?also is known as river prawn?is an important shrimp aquaculture species.Because of its' good adaptability and strong reproductive strength,river prawn has been widely distributing in China and are widely welcomed by the Chinese market.However,with the development of modern industrialization,coupled with the unhealthy and irregular development of the aquaculture industry,the water quality has been in different degrees of destruction,and the shrimp farming suffering from a variety of diseases.However,the study of river prawn's innate immune defense mechanism can guide the production in practice to carry out disease prevention and control strategy effectively.The important components of innate immunity of invertebrates are various kinds of immune signaling pathways and the pattern recognition receptor.In this paper,the Mats gene in the Hippo signaling pathway of Macrobrachium nipponense is studied firstly.Yorkie is a transcription factor in Hippo signaling pathway,which promotes the expression of Cactus gene?Toll signaling pathway?.Yorkie,as a transcription factor,needs to be interoperable with other proteins,Taiman is a kind of mutual protein of Yokie,so we make a preliminary study on the selective splicing of Taiman and its function in innate immunity defense.Finally,we make a preliminary study on the function of a highly variable C-type lectin?To increase the diversity of lectin by containing different numbers of repetitive units in the coding area.?.1.The function of Mn Mats gene in innate immunity of Macrobrachium nipponenseWe found and named the Mn Mats gene,its c DNA length is 1127 bp and open reading frame?ORF?size of 651 bp encoding 217 amino acids length of the protein,which contained a Mob1_phocein domain.Tissue distribution in river prawns shows that it was distributed in various tissues with the largest expression in the hepatopancreas and the smallest in hemocytes.The further study focus on the expression pattern after Staphylococcus aureus/Vibrio parahaemolyticus stimulation,the result shows that after Staphylococcus aureus stimulation each tissue's Mn Mats showed a trend of rising in addition to hemolymph and stomach.And when the host in the face of Vibrio parahaemolyticus challenge,in addition to the stomach,others also increased.After RNAi knocked down,it was found that a variety of antimicrobial peptides has a lower expressing level compared to the control group.These results show that,as an important component of the Hippo signaling pathway,The Mn Mats gene may promote the expression of antimicrobial peptides by inhibiting the expression of Cactus gene,thus participating in the innate immune defense of Macrobrachium nipponense.2.The function of Mn Taiman gene in innate immunity of Macrobrachium nipponenseWe found and named the Mn Taiman gene in Japanese prawns.There are four different transcription copies of the Taiman gene of Macrobrachium nipponense.The longest transcription of this c DNA is 6894 bp,the open reading frame?ORF?is 4998 bp coding a protein with the length of 1666 amino acids which contains a PAS domain,a HLH domain,and a PAS₁1 structure domain.Semi-quantitative tissue distribution found that selective shear was present at the C terminal of Mn Taiman gene,resulting in four different shear copies,named Tai A,Tai B,Tai C and Tai D.The results showed that the tissues distribution of Mn Tai-Total?Tai T?,Tai A,Tai B,and Tai D were similar in q RT-PCR,distributed in various tissues,and highly expressed in the hepatopancreas.while Tai C is highly expressed in stomach.The further study focus on the expression pattern after Staphylococcus aureus/Vibrio parahaemolyticus stimulation,the result shows that,in addition to hemocytes,Tai T and the four transcriptions were presented in different expressions.In the hymocytes,the genes showed a downward trend after the pathogen stimulation.In the hymocytes,while knock-down of Mn Taiman,the expression of ALF1 increased significantly after 24 h past the stimulation of Staphylococcus aureus.The expression of ALF1 was also significantly increased after 24 h past the stimulation of Vibrio parahaemolyticus.According to the existing papers,Taiman and Yorkie work with each other,while Yorkie regulate the expression of Cactus.Therefore,we speculate that Mn Taiman inhibits the expression of antimicrobial peptide genes by regulating the expression of Cactus and negatively regulating the Toll signaling pathway.3.Study on the immune function of a hypervariable C-type lectin Mn RlecWe found a type of C-type lectin in Macrobrachium nipponense,and because its encoding area contains a different number of repeating units,we named it repeat lectin-Mn Rlec.It contains a signaling peptide,a CLECT domain and an area between the two domains containing a different number of repeating units,which can contain up to 9 series repetitions and at least one series repetition.On the genome,we have also found sequences containing different numbers of repeating units,so we speculate that these repetitive units can have a sliding mismatch during genomic replication,leading to the production of genetic diversity.We expressed and purified three kinds of Mn Rlec proteins containing 1,2 and 3 repeating units,named Mn Rlec1,Mn Rlec2 and Mn Rlec3.The bacterial binding experiment of these three proteins showed that the combination of Mn Rlec1 on Vibrio parahaemolyticus was Ca2+ dependence,and the binding ability of Mn Rlec2 and Mn Rlec3 on Vibrio parahaemolyticus decreased.In addition,Mn Rlec1,Mn Rlec2 and Mn Rlec3 all exhibit the ability to bind Escherichia coli,and this combination does not depend on Ca2+.Through our study,it was found that the Mn Rlec gene weakened with the increase of repetitive units and the ability to combine Vibrio parahaemolyticus,which may be the result of a game between the pathogen and the host during evolution.