Construction Of Lotus Japonicus AD-cDNA Library And Identification Of Proteins Interact With Nod Factor Receptor NFR1

The rhizobia develop root nodules with legumes, the symbiotic relationship is specified. The plant supply carbon resource for the rhizobium by photosynthesis, the rhizobium supply nitrogen resource for the plant by reducing the nitrogen from air through the bacterial nitrogenase. The rhizobial symbiosis is initiated by flavonoid secreted by the plants, when the flavonoid is perceived by rhizobia the nod factor is released from rhizobia, the nod factor is the rhizobial signal inducing root hair deformation and cell division leading to development of root nodule. Nod factor receptor 1(NFR1) is a gene acquired from mutated Lotusjaponicus that can not form nodules. It's believed that NFR1 is responsible for the perception of nod factor and the signal transduction. The amino-terminal region of NFR1 consists of two LysM motifs which are directly related to the perception of nod factor. A transmembrane segment anchors the receptor to the plasmid membrane. The carboxy terminus of the NFR1 protein contains a functional serine/threonine kinase. It's believed that the kinase domain of NFR1 transfers the signal by phosphatizing other molecules. But the molecules interact with NFR1 is still unknown. The yeast two hybrid system is developed to detect the interaction of two proteins in the recent years. To determine the specific role of NFR1 in the signal transduction pathway and explore the molecules in this cascade we constructed a AD-cDNA library, and screened the library by yeast two hybrid using NFR1 as the bait protein, provide some candidates for the NFR1's signal transduction partner.Cultivate Lotus japonicus for the plant tissue; harvest the root of Lotus japonicus after inoculation by 2d, 4d, 6d, 8d and 12d. Combine all the tissues and extract the total RNA. Synthesize the ds cDNA by RT-PCR, cotransform the yeast strain AH109 with ds cDNA and pGADT7-Rec, the recombination plasmid will express the cDNA insert as a GAL4 AD fusion protein. Select transformants on SD/-Leu plates, harvest tranformants into Freezing Medium, store the library as 1 ml aliquot at -70℃.The transformation efficiency is 2×10~6 which is calculated by diluted plates, exceed the expected 1×10~6 transformants/3μg pGADT7-Rec. The PCR result of random chosen library colonies shows that the average length of cDNA inserts is 1.5 kb, the colonies without cDNA insert is 12.5% of the total. The cell density of library is 2×10~7/ml.The kinase domain of NFR1 is cloned to the pGBKT7 as the bait plasmid; transform the bait plasmid to the yeast strain AH109. Mate the library strain with the bait strain. Select the yeast diploids expressing interacting proteins on quadruple dropout medium. We got 120 Ade~+, His~+ colonies and restreak the colonies on quadruple dropout medium containing X-gal, chose the colonies turn blue. Isolate AD plasmids form positive colonies; amplify the cDNA insert by PCR. Retest the positive colonies by cotransforming the AD plasmids and bait plasmid to AH109. Sequence the cDNA inserts and we got several genes. To determine the expression of those genes, half-quantitative RT-PCR is performed. Those gene provide candidates for the NFR1's signal transduetion partner.