Cloning And Expression Analysis Of Meloidogyne Javanica Mj-TTL6 And Mj-TTL8 Genes

Meloidogyne javanica is one of the most common root-knot nematode in China and can attack more than 2,000 plant species.It is widely distributed in Southern China,causing serious yield losses to crops.M.javanica would secrete a variety of effectors into host plants during parasitism.Identifying and characterizing effectors from M.javanica can help understand the molecular mechanism of M.javanica pathogenicity,which provide a theoretical basis for the control of M.javanica.In this study,two Mj-TTL genes were successfully cloned from M.javanica by homologous cloning and SMART RACE methods,and their cDNA and DNA sequences were obtained.The in situ hybridization,qRT-PCR and yeast two-hybrid methods were used to analyze their functions.The main results are as follows:1.Two transthyretin-like protein genes from M.javanica were cloned.The length of Mj-TTL6 ORF is 444 bp,the length of Mj-TTL6 DNA is 1184 bp,interrupted by two intron.The Mj-TTL6 is predicted to encode a protein containing 148 amino acid.Blast using Mj-TTL6 in EST library of NCBI showed the highest homology with a M.incognita EST(97%).The BLASTx alignment analysis showed the highest homology with TTL of Toxocara canis(58.7%).The length of Mj-TTL8 ORF is 423 bp,the length of Mj-TTL8 DNA is 907 bp,interrupted by two intron.The Mj-TTL8 is predicted to encode a protein containing 141 amino acid.Blast using Mj-TTL8 in EST library of NCBI showed the highest homology with a EST of M.areanaria(98%).The BLASTx alignment analysis showed the highest homology with TTL of Onchocerca volvulus(59.6%).Both proteins have an N-terminal signal peptide and a TTL conserved domain,TTR-52.Both proteins do not contain transmembrane structures.2.A phylogenetic tree from Bayesian analysis of TTL was constructed.The tree supported the monophylies of the different TTL proteins with high support.Of these,Mj-TTL6 and TTL6 from M.incognita and M.areanaria were positioned in the same clade with 100% supported,and Mj-TTL8 clustered with other five TTL8 protein of root-knot nematode with 100% supported.The result show that different TTL proteins may have different functions.3.In situ hybridization demonstrated that Mj-TTL6 is specifically expressed in the dorsal esophagus glands of M.javanica pre-parasitic second stage juveniles(pre-J2),and Mj-TTL8 is specifically expressed in the subventral gland of M.javanica pre-J2.4.qRT-PCR showed that the expression level of Mj-TTL6 was very low in the egg phase of M.javanica,increased obviously in pre-J2,then decreased significantly after infection,and then increased steadily,reaching the maximum expression in mature female stages.It showed that Mj-TTL6 has a specific role in the pre-J2 and female.The expression level of Mj-TTL8 was low in both egg and pre-J2,increased gradually,and reached the maximum expression in mature female stages.It showed that Mj-TTL8 has a specific role in the late stage of parasitism.5.Yeast two-hybrid experiments revealed that Mj-TTL6 can interact with Arabidopsis thaliana protein encoded by chloroplas and glycoside hydrolase second family protein,suggesting that Mj-TTL6 may promote parasitism by regulating the development of host plants or the immune system.Mj-TTL8 can interact with Arabidopsis thaliana FK506-binding protein 12,suggesting Mj-TTL8 may regulate DNA content in plant cell to promote parasitism of nematodes.In summary,Mj-TTL6 and Mj-TTL8 are two novel secretory effectors.Mj-TTL6 plays a role in the pre-J2 and the late stage of nematode parasitism,and Mj-TTL8 plays a role in the late stage of nematode parasitism.