Functional Verification Of Several Nitrogen-fixing Genes In Anabaena Sp. PCC 7120

Anabaena sp. PCC 7120 is a typical model organism in the study of bacterial phylogeny and multicellular structure. In the absence of compound nitrogen in the environment, there is some vegetative cells differentiate into heterocyst. The main contents were shown as follows: Based on our laboratory studies,selecting the genes which may be related to nitrogen fixation, Using p ZR606 knock one target gene, insert part of the target gene sequence into the MCS of p ZR606, use single-crossover homologous recombination knocking target gene and purifing knockout strain of Anabaena PCC 7120 by colony PCR, observe the change of knockout strain on the lack of compound nitrogen medium, identify the target gene which is related to nitrogen fixation; pZR670 has a strong promoter which called Pgln A was selected as complementary plasmid, observe whether the complementary strain recovery after covering a certain gene expression in the lack of compound nitrogen; By analyzing the transcriptome data of Anabaena PCC 7120, choose sRNA which may has a different in phenotypic changes, pZR670 aslo selected as interference plasmid, target plasmid transcribed out of the complementary sequence making the target sRNA in silencing or loss of it,s function. Then, observating the changes.The major results were shown as below:Choose Anabaena PCC 7120 cells when growth well were seeded in AA/8 and AA/8N liquid culture, In AA/8 or AA/8N liquid culture conditions, detecting the absorbance value in 700 nm of the wild type Anabaena PCC 7120, found nitrogen not only a limiting factor for cell growth, but also the key element in heterocyst formation.Successful use of single-crossover gene recombination technology, which based on p ZR606 plasmid as a basic skeleton, knockout the all2022, all2676, alr0834 gene and get the knockout plasimd pHEP52, pHEP34, pHEP55 and the konckout strains ?all2022, ?all2676, ?alr0834, when cultured a period of time on AA medium,which not containing nitrogen source, the knockout strains will turn yellow.Successful use pZR670 constructed all2022, all2676 and alr0834 complementary plasmids pHEP90, pHEP91, pHEP92 and complementary strain ?all2022, ?all2676, ?alr0834, the complementary strain ?all2022 not resume normal growth, but ?all2676 and ?alr0834 can grow well in mediun which not fixed nitrogen source, so,all2676 and alr0834 are related to fixing Nitrogen, all2022 probably may be an intermediate gene of one operon leading to cell death effects of polar effect.Successful use pZR670 as a backbone to construate interference sRNA plasmids and interference strains, the strain were cultured on the AA/16 N and AA/16 solid medium, or in the AA/8N liquid medium, compared to changes, found strain which containing plasmid can be transcribed sequence, which include the complementary sequence of sRNA0425, be easy to growth of adherent in AA/8N liquid; the strain which containing plasmid can be transcribed sequence, which include the complementary sequence of sRNA1064, the number of cells in filaments can not more than 30.