Cloning Of Calmodulin Gene Promoters In Lotus Japonicus And Analysis Of Their Activities In Nodule

In order to increase the yield of crops,applying chemical fertilizer is the most direct and effective method,but this will increase the burden on environment.For example,the soil nutrition structure is damaged.The soil is hard.And soil erosion is intensified.The legumes represented by Lotus japonicus are often used as green manure.Because of its strong nitrogen fixation,it can effectively improve the soil,increase the nitrogen fertilizer content in soil,reduce the use of chemical fertilizer,and protect the environment while ensuring production.Therefore,the research on the biological nitrogen fixation of legumes has certain significance.Some achievements have been made on the molecular mechanism of nitrogen fixation symbiosis.Many genes involved in this process have been cloned and analyzed including calcium/calmodulin-dependent protein kinase（CCa MK）,which is an important signaling factor involved in the regulation of rhizobia and mycorrhizal symbiosis in leguminous plants.Its activity is regulated by both Ca²⁺and calmodulin（Ca M）,suggesting that Ca M may also be involved in the process of nitrogen fixation symbiosis.Promoter plays an important role in the regulation of gene expression.The analysis of promoter sequences can effectively predict the possible biological functions of genes.It is also of certain guiding significance to study the function of the gene by exploring the activity of promoter.Therefore,in this study,L.japonicus was used as the material,and the promoter sequences of LjCaM1,LjCaM2,and LjCaM4 were amplified by chromosomal walking technology;the cis-acting elements of them were analyzed by bioinformatics;Constructing the vectors of p BI101-p LjCaM1-GUS,p BI101-p LjCaM2-GUS and p BI101-p LjCaM4-GUS,and the recombinant plasmids within Agrobacterium tumefaciens LBA4404 were transformed into Nicotiana benthamiana to explore promoter activity.These three recombinant plasmids within Agrobacterium rhizogenes AR1193 were transformed into Gifu plants,and then the transgenic plants were stained byβ-glucosidase staining,and the activities of GUS in roots and nodules was observed under microscope.The main experimental results are as follows:（1）LjCaM1 gene sequence was 1679 bp,including 2 exons and 1 intron.The exons were 75 bp and 375 bp,respectively.The intron was 1229 bp.（2）LjCaM1 promoter sequence is 1122 bp;LjCaM2 promoter sequence is 2077 bp and LjCaM4 promoter sequence is 2090 bp;（3）Through bioinformatics analysis,there are 2,3,and 4 transcription start sites greater than 90%on the promoter sequence of LjCaM1,LjCaM2,and LjCaM4,respectively;There are also some differences in the types of cis-acting elements between them.p LjCaM1 mainly has response elements related to plant immunity,p LjCaM2 has many light-responsive cis-acting elements,and p LjCaM4 has cis-acting elements that respond to abscisic acid;（4）the amplified promoters of LjCaM1,LjCaM2 and LjCaM4 can drive GUS expression.（5）Observing the hairy roots transformation plants stained withβ-glucuronidase,the activity of LjCaM1,LjCaM2,and LjCaM4 promoters were different.Both LjCaM2 and LjCaM4 promoter activities can be detected in apical meristem,and LjCaM4 promoter activity is stronger than that of LjCaM2.LjCaM2 promoter activity can also be detected weak activity in root vascular bundles.The activity of LjCaM1 promoter cannot be detected in fully mature root nodules,except for weak activity in root nodule parenchyma cells and root nodule vascular bundles;LjCaM2 promoter only weakly detected in root nodule vascular bundles;The activity of LjCaM4 promoter was mainly detected in the nodule parenchyma cells and root nodule vascular bundles,but the center cells infected by bacteria was not detected.In this experiment,through bioinformatics analysis and observation of plant tissue sections,it was found that LjCaM1,LjCaM2 and LjCaM4 had certain similarities in function and expression sites.All of them can detect their promoter activity in nodule vascular bundles,indicating that they may be involved in the transport of root nutrients.While LjCaM2 promoter contains a lot of light response of cis element,explain LjCaM2probably by light regulation to influence the root nutrient transport,LjCaM4 promoter contains response abscisic acid used by cis element,and in the parenchyma cells in the root nodules can be detected LjCaM4 promoter activity,suggests that it may adjust action on nodule development by abscisic acid.