Identification And Expression Analysis Of Sorghum 14-3-3 Gene Family And Screening Of Crystal Growth Conditions

Sorghum [Sorghum bicolor(L.)Moench] is an important economic cereal.It has strong growth ability,strong salt and alkali resistance,drought resistance,high temperature resistance and other stress resistant characteristics.However,various adverse environmental factors(biological stress and abiotic stress)have a great impact on the yield and quality of sorghum.Therefore,it is significant to carry out the important resistance genes in sorghum.14-3-3 protein,as a conserved protein widely existing in eukaryotes,plays an important role in the resistance of plants to various adversities.In this study,bioinformatics methods were used to identify the 14-3-3 gene family in sorghum,and the basic physical and chemical properties of amino acids,conserved domains,motifs,and phylogeny were analyzed.Subsequently,real-time fluorescent quantitative PCR(q RT-PCR)was used to analyze the expression profile of the 14-3-3 gene family in response to abiotic and biotic stress.Three genes were successfully cloned and their prokaryotic expression conditions were explored.Finally,the protein purification system was used to purify the above-cloned protein,and the crystal growth conditions were screened by the hanging drop method.The results are as follows:1)Using local BLASTp and Hmmer methods,six 14-3-3 family members were identified in sorghum and named SbGF14a-SbGF14f.The protein sequence coded by this family member is between 247-266 Aa in length,the protein molecular weight is about 28 k Da,and the theoretical isoelectric point is 4.76 to 5.17,slightly acidic,contains multiple conservative motifs,and is closely related to maize.2)Analyzing the tissue specificity,response to abiotic stress(salt,drought and plant hormones)and biotic stress(PAMPs)of the 14-3-3 gene family in sorghum,the results indicate that the 14-3-3 genes are tissue specific.Besides,drought stress,salt stress,PAMPs,plant hormones could induce the expression of its members.3)The SbGF14c,SbGF14d and SbGF14f genes were cloned,and they were successfully constructed into the prokaryotic expression vector p ET-28 a,and the protein expression conditions such as the expression strain,induction temperature and IPTG induction concentration were studied.Finally,the optimal conditions for the soluble expression of the above-mentioned protein were successfully obtained.4)SbGF14c,SbGF14d and SbGF14f proteins were purified by using AKTA Pure Protein Purification System,and the obtained proteins were with higher concentration and purity.5)The crystal growth conditions of SbGF14c,SbGF14d and SbGF14f proteins were screened by using the hanging drop method.The protein crystals can be observed in Index-96,Salt Rx1-28;PEG/Ion-1,PEG/Ion-2 and Index-39,Index-15 conditions,respectively.Finally,the obtained crystal was diffracted by an X-ray single crystal diffractometer,and the diffraction results demonstrate that they could be protein crystals.