Preliminary Study On BnaLCR78 Gene In Brassica Napus L.

The development of rapeseed and the anabolism of oil involve a complex network regulation system.In this study,a functionally unknown gene expresses in the late development of rapeseed,homologous to the Arabidopsis LCR family,which is named BnaLCR78 has been studied by means of bioinformatics,molecular biology and molecular genetics.The results are shown as follows:1.Bioinformatics analysis revealed that there are two copies of BnaLCR78,BnaLCR78.A and BnaLCR78.C in Brassica napus,respectively.BnaLCR78.A gene is 531 bp in length and BnaLCR78.C gene is 538 bp in length.The two genes contains the same CDS sequence:a total of 237 bp,encoding 78 amino acids.The translated protein contains a signal peptide and a transmembrane domain and so it is presumed to be a secreted protein.,and contains a transmembrane domain,.2.The spatiotemporal expression analysis of BnaLCR78 showed that it was undetectable in roots,stems,leaves and flowers while trace amount in the pods and stable in seeds.Further histochemical staining analysis revealed that the BnaLCR78 gene was specifically expressed in the cotyledons of the seeds.3.The following vectors were constructed:constitutive promoter 35S and seed-specific promoter napin-activated overexpression vector 35S::BnaLCR78,Pnapin::BnaLCR78;interference vector 35S::BnaLCR78RNAi,Pnapin::BnaLCR78RNAi;knockout vectors sgRNA-Cas9-78 and pYLCRISPR/Cas9-BnaLCR78.4.The constructed vectors were transformed into the hypocotyl of"XY15"in Brassica napus by agrobacterium-mediated transformation.Follwed by molecular detection of resistant seedlings:8 transgenic plants were successfully obtained for 35S overexpressing with the transformation rate of 2%;19 transgenic plants for Napin overexpressing with the transformation rate of 2.38%;12 transgenic plants for 35SRNA interfered with the transformation rate of 3%;21 transgenic plants for napin interfered with the transformation rate of 4.67%;26 transgenic plants for pYLCRISPR/Cas9-BnaLCR78 with the transformation rate of 2.17%;26 transgenic plants for sgRNA-Cas9-78 with the transformation rate of 7.9%.5.The homozygous mutation of knockout transgenic plants were successfully obtained in T₀ generation.The editing efficiency of transgenic sgRNA-Cas9-78 vector was 70%and the edit form is mainly a single base insertion.