Effect Of Histone Deacetylase Gene LjHDT2(L. Japonicus) On The Symbiotic Regulation Of Arbuscular Mycorrhizal Fungi

Arbuscular mycorrhizal fungi(AMF),as a kind of beneficial microbes,can build a symbiotic relationship with 90% vascular plants.Studies had shown that Lotus japonicus is an important model of leguminous plants and can form a symbiotic relationship with AMF,which not only enhances plant resistance,but also has a good application value in agriculture,forestry,and animal husbandry.Numerous studies had reported that histone deacetylases(HDAC)can affect the modification of the amino residues of histones,and the downstream genes can also be influence by HDAC too.Currently,the researches on this gene family are mainly focused on the growth and development of model plants such as Arabidopsis thaliana and rice,but there are no reports on how to regulate the symbiotic signal transduction pathway between plants and microorganisms.Therefore,encoding the HDACs' gene of L.japonicus was selected by using bioinformatics technology.qRT-PCR analysis showed that LjHDT2 could be significantly induced by AMF,in order to analyze the effect of this gene on AMF symbiosis,RNAi technology was used to knock-down the LjHDT2.The positive plant(LjHDT2-RNAi transgenic L.japonicus)was obtained.According to the infection efficiency of AMF,preliminarily revealed the roles of LjHDT2 in the symbiotic system of AMF and L.japonicus.The results are shown as follows:1.The target gene is a member of the HD2 family,and this gene was always good for the symbiosis relationship established.qRT-PCR results showed that AMF significantly induced the expression level of LjHDT2 in L.japonicus.Sequence homology comparison also found that the gene has high similarity with the MtHDT2 which are the HD2 family of Medicago Sativa,from this we named it LjHDT2.2.Sequence analysis showed that the LjHDT2 gene's length is 1156 bp,and it can also encodes 297 amino acids.The molecular mass of the protein is 32.12 kD,and the isoelectric point is 4.88.By subcellular localization predictions,the result indicate that the protein of LjHDT2 is localized in the nucleus.3.The LjHDT2-RNAi interference recombination vector was constructed successfully,the recombinant plasmid was also introduced into the L.japonicus by Agrobacterium rhizogenes.LjHDT2-RNAi transgenic L.japonicus was obtained.Quantitative PCR analysis of LjHDT2 gene expression in transgenic L.japonicus revealed that the gene was significantly downregulated.4.After the interaction analysis with AMF found that,when silencing LjHDT2,the time required for AMF colonization in transgenic L.japonicus becomes longer,and it takes about 20-25 days to establish a stable symbiosis and produce spores,the control plants only took 15-20 days.It was also found that the colonization rate of AMF in transgenic L.japonicus was decreased,and the colinization rate of spores in both was also differently.After 15-30 days of infection,the colonization efficiency of AMF was 16.67%-33.05%,on the other hand,the AMF colonization rate of the wild type L.japonicus' root was 23.50%-44.44%.The ratio of AMF colonization at 30 days in transgenic L.japonicus were not significantly different with the control plant at the 20 days.The root number is also no significant difference between transgenic L.japonicus and wild type L.japonicus.