Molecular Evolution And Functional In Nodulation Analysis Of Lysine-Histidine-Like Transporter Genes In Glycine Max(Soybean)

Nitrogen is one of the most important nutrients for plant growth and development,and accounts for about 5%of the dry weight in plant,Nitrogen exists mostly in the form of amino acid polypeptides and proteins and participates in the life process of organisms.Soybean is an important economic and cultivated crop,Its seeds are rich in protein and contain essential amino acids which we must get from our diets.Soybean and Rhizobium establish symbiotic nitrogen fixation system to help plants convert nitrogen gas,which help plants to convert nitrogen in the air that cannot be directly absorbed and utilized by plants into nitrogen resources that can be absorbed by plants Amino acid transporters play an important role in the transport,distribution and absorption of various amino acids and in response to biological and abiotic stresses in plants.There are few reports about the role of amino acid transporters in the regulation network of nodulation and nitrogen fixation in Soybean.In this study,the evolutionary mechanism of LHT gene family in Angiosperm was analyzed through the research methods of bioinformatics and molecular biology using nine representative plants with genome-wide data,and the functional differentiation of LHT gene family in soybean was also discussed.The nodulation and nitrogen fixation pathway of Gm LHT gene was preliminarily explored by genetic transformation of Lotus corniculatus L.and subcellular localization technology.The main results are as follows:(1)In 9 representative species,we identified a total of 114 LHTs,There are 25 in Soybean,and the number of introns was 1-9.Based on multiple sequence alignment and phylogenetic tree analysis,we divided the 114 LHTs into I and VII groups,group II has the most members,and group III is specific to the monocot plants;The number of amino acids encoded in soybean was 56-543aa,the relative molecular weight was(6.3-57.1)×10~3,and the theoretical isoelectric point was 6.03-9.51,all members were transmembrane proteins;(2)By mapping Gm LHTs on chromosomes and collinearity analysis,we identified 8paralogs,which indicated that 64%of the homologous copies in Gma LHT gene family were the result of WGD or tandem duplication.It suggests that the LHT gene family has been expanding continuously during plant evolution,and its expansion is closely related to genome repetition.Through the estimation of Ka/Ks of 8 pairs of paralogitic homologous genes,it was found that Ka/Ks were all less than 1,indicating that strong purification selection played a key role in the differentiation of gene function after whole-genome replication.(3)Expression pattern analysis showed that the expression of Gma LHTs can be divided into two categories:one is the expression of some tissue specific genes,the other is the genes that were low expression levels in all detected tissues.The expression of this gene family was induced by rhizobium infection and the specific expression of some members in root and root tumors after root infection.The differential gene expression patterns of LHTs in different evolutionary groups in soybean suggested that it maybe undergone by selection in the process of evolution,and the transmembrane domain plays the important role in the integrity of its functions.(4)The expression pattern of Gma LHTs gene in soybean was analyzed by q RT-PCR.The results showed that Gma LHT17 was highly expressed in root nodules,which may play an important role in nitrogen fixation in soybean nodules;Gma LHT18was highly expressed in the soybean leaves inoculated with rhizobia,which may play an important role in plant growth.However,the expression of Gma LHT24 in all tissues was low.(5)Predicting the functions of proteins,it was found that alpha-helix was the main component of proteins in Gma LHT17、Gma LHT18 and Gma LHT24,all of which were hydrophobic proteins and had no signal peptide structure.Gma LHT18 and Gma LHT24contained more phosphorylation sites.Plant-Ploc predicts that Gma LHT17、Gma LHT18 and Gma LHT24 may be located on the plasma membrane,endoplasmic reticulum and mitochondrial membrane in order to perform their transport functions.(6)Agrobacterium tumefaciens-mediated transformation technology was used to induce rooting in Lotus corniculatus L.to discuss it’s effect on soybean nodulation.According to the statistics of nodulation number of transgenic plants,we found that overexpression of Gma LHT17 and Gma LHT18 could significantly increase the number of nodulation;however,overexpression of Gma LHT24 could significantly reduce the number of nodules in root hairs of Lotus corniculatus L..It is speculated that these genes may be involved in some regulatory pathways,thus affecting the regulation network of nodulation and nitrogen fixation,and further affecting the nodulation ability.(7)The expression vector of Gm LHTs and e YFP was constructed.Onion epidermal cells were infected by Agrobacterium tumefaciens.The localization of Gma LHTs in onion epidermal cells was observed under fluorescence microscope.Gma LHT17 and Gma LHT24 were transformed into onion epidermal cells by Agrobacterium tumefaciens.The location of Gma LHT17 and Gma LHT24 was observed under fluorescence microscope,and found the location of Gma LHT17 and Gma LHT24was plasma membrane,we speculated that amino acids are transported into/out of the cell by the cytoplasmic membrane.In summary,our results can serve as an important foundament for further research of soybean LHT gene family,and also provide new ideas for the study of amino acid transporters.The above results provided important genetic information and theoretical basis for the future development of targeted modified soybean amino acid transport capacity and improving the nitrogen fixation efficiency of soybean.