Cloning Of BnaC03g45680D Gene From Brassica Napus And Construction Of Plant Expression Vector

Brassica napus has become an indispensable oil crop in our life.It is of great academic and practical significance to analyze and excavate important functional genes of Brassica napus.Secreted protein is a type of protein that acts on different parts after being synthesized in the cell and then released outside the cell,and serves as a bridge between cell protoplasts and the environment.In plants,proteins mainly include plant intercellular fluid and root exudates.They are important for cell growth,metabolism and stress resistance.In the study of Brassica napus resistance to the environment,the intercellular liquid protein of Brassica napus leaves,which responds to low phosphorus stress,was encoded by the Brassica napus gene BnaC03g45680 D,but the biochemical and physiological functions of the Brassica napus are rarely reported.Based on the previous work,the homology of BnaC03g45680 D protein was compared and analyzed,and the phylogenetic tree was constructed,and the related bioinformatics analysis was carried out.Taking the Brassica napus "Ding oil zaja No.4" as the test material,the CDS region of gene BnaC03g45680 D was amplified by RT-PCR technology to construct cloning vector and plant expression vector.The recombinant vector was introduced into the Agrobacterium tumefaciens GV3101 by freezing and thawing method,which laid the foundation for the later using Agrobacterium mediated method to transfer the gene to rape and other plants.The main results obtained are as follows:1.Using CDD online analysis gene BnaC03g45680 D encoding protein with Basic secretory protease(BSP)conservative domain,Through alignment of BLASTP homology,11 amino acid sequences with higher homology between different species were selected,and a phylogenetic tree was constructed using the neighboring method(NJ)of MEGEA 6.Result show that the radish,hawthorn,leeks,flax pods and Arabidopsis proteins are closely related to the BnaC03g45680 D protein in Brassica napus.2.According to the physicochemical properties of the ExPASy Prot Param prediction gene BnaC03g45680 D encoding protein,it is found that the protein contains 225 amino acids,the molecular weight is 24997.90 Da,the isoelectric point is 6.20,and the instability coefficient is 29.88,so it is stable.The aliphatic amino acid index was 72.71 and the protein hydrophilicity was-0.333.Using the online software Hphob./Kyte&Doolittle model to analyze the pro/hydrophobicity,the results showed that the number of positive peaks was significantly greater than the number of negative peaks,indicating that hydrophilicity dominated and was soluble protein.3.Using the signal peptide location of the SignalP 4.1 Serve online prediction gene BnaC03g45680 D encoding protein,The results show that the first to 22 amino acids may be signal peptide regions,and there is a shear site between twenty-second and twenty-third amino acids.Subcellular location prediction of gene BnaC03g45680 D encoding protein showed that the extracellular matrix and endoplasmic reticulum were the most likely,followed by vacuoles.4.The secondary structure prediction of gene BnaC03g45680 D encoding protein showed that its main structural elements were ?-helices and random coils,as well as extended chains and ?-turns.The phosphorylation site of gene BnaC03g45680 D encoding protein was predicted and found to have eight Ser phosphorylation sites,one Thr phosphorylation site,and two Tyr phosphorylation sites.Thirteen N-glycosylation sites were predicted by NetNGlyc 1.0 Server.5.Total RNA was extracted from leaves of Brassica napus(Dingyouza No.4)and reverse transcribed into cDNA.A pair of primers was designed against the coding region of BnaC03g45680 D gene and a gene fragment 678 bp was amplified by RT-PCR.The target fragment was ligated into the linearized vector pBM26 and the sequence similarity to the published sequence in the Genbank library was 100%.6.On this basis,the plant expression vector PCAMBIA1300S-BnaC03g45680 D containing the 2×CaMV35S promoter was constructed.After colony PCR,restriction enzyme digestion and sequencing,the insertion sequence was the target fragment.The recombinant vector was introduced into Agrobacterium tumefaciens GV3101 by freeze-thawing method,which laid a good research foundation for later use of Agrobacterium vector method to transduce BnaC03g45680 D gene into Brassica napus and other plants and explore its physiological and biochemical functions.