The Effects On The Growth Of Medicago Truncatula Nodules Of Knocking Out Genes Specifically Expressed In Nodules

Medicago truncatula s is an annual leguminous herb with small ploidy,small genome,self-pollination and more seeds.As a model plant,Medicago truncatula has been widely used in laboratories because of its short plant regeneration time and high genetic transformation efficiency.The symbiotic nitrogen fixation system formed by leguminous plants and Rhizobium is one of the most important ways of nitrogen fixation in nature.Leguminous plants supply carbohydrates of rhizobium,and Rhizobium provides nitrogen nutrients of plants,thus forming a mutually beneficial symbiotic relationship.Its essence is a very complex behavior of molecular dialogue,which involves a series of gene regulation and expression processes in leguminous plants.Therefore,it is of great significance to study the expression and regulation of genes related to nodulation and nitrogen fixation in nodules.Previously,RNA-Seq was used to study the nodule roots of Medicago truncatula.It was found that Medtr3g079850 and Medtr8g089360 were not expressed in hairy roots,but only in nodules.It is speculated that it plays an important regulatory role in the nodulation process and may have an impact on the symbiotic nitrogen fixation of Medicago truncatula.Through sequence alignment analysis,The Medtr3g079850 gene is presumed to come from the receptor-like protein kinase family.Medtr8g089360 gene is presumed to come from cationic amino acid transporter family.In order to further explore the effect of above genes on the nodulation process of Medicago truncatula,we used CRISPR/Cas9 gene editing technology to knock out the selected genes and observe their effects on the nodulation process of Medicago truncatula.Through qRT-pcr,we studied the effect of these genes on the nodulation process of Medicago truncatula.The dynamic expression characteristics of the target gene in the nodulation process of alfalfa were studied by PCR,and the expression sites of the selected gene translation products in the nodules were determined by GUS staining.The results will lay a good foundation for exploring the specific functions of the above genes in the symbiotic interaction between Medicago truncatula and rhizobium.The main contents and results of this experiment are as follows:CRISPR/Cas9 gene knockout vectors of Medtr3g079850 and Medtr8g089360 genes were constructed.The constructed CRISPR/Cas9 gene knockout vectors were introduced into Medicago truncatula by Agrobacterium Rhizogenes-Mediated plant receptor transformation technology.After inoculating with rhizobium,the effects of CRISPR/Cas9 gene knockout vectors on the growth,development and structure of Medicago truncatula Rhizobium were observed.The results showed that when Medtr3g079850 and Medtr8g089360 genes were knocked out,the number of lateral roots and the length of hairy roots of transformed plants were significantly reduced,the nodulation ability was weakened,the number of nodules was reduced,the number of effective nodules was reduced,and the nodules were small and white.The dynamic expression characteristics of the target gene during the growth of nodules in Medicago truncatula were studied by qRT-PCR.It was found that Medtr3g079850 was mainly expressed in the early stage of nodulation,and Medtr8g089360 was expressed in the whole process of nodule development.The GUS expression vector with Medtr8g089360 gene promoter was constructed.The expression site of the selected gene translation products in nodules was determined by GUS staining.The results showed that the Medtr8g089360 gene translation products were mainly located in the infected area.