| Sorghum grain has been used as food,feed and stocks for food processing industry.Improvement of grain quality is an important goal in sorghumbreeding.Sorghum grain has a variety of functions and USES.Sorghum alcohol soluble protein,or kafirin,is a seed storage protein,which has great application potential in food and industry.The kafirin gene was identified by bioinformatics,and its expression,DNA variation and phylogenetic analysis were carried out.By optimizing the parameters of sorghum tissue culture,a regeneration system suitable for sorghum genetic transformation was established to provide a basis for further genetic improvement of sorghum.The results show that:1.In the sorghum genome 24 genes were identified,namely,alpha-kafirin,beta-kafirin,gamma-kafirin and delta-kafirin.Alpha,beta,gamma,delta kafirin protein secondary structure is mainly alpha helix and random curl,is a hydrophobic protein,signal peptide.The subcellular localization is membrane binding protein.The isoelectric point PI of the alpha-kafirin theory is 6.36-9.25,and the molecular weight is 24873.84-31595.47 Da,which is an unstable protein.Beta-kafirin theory is an unstable basic protein with an isoelectric point PI of 7.99 and molecular weight of 20768.64 Da.Gamma-kafirin has a transmembrane spiral domain,the theoretical isoelectric point PI is 8.20,and the molecular weight is 22059.75 Da,which is an unstable basic protein.Delta kafirin theory is an unstable basic protein with an isoelectric point PI of 8.77 and a molecular weight of 16516.31 Da.2.Alpha-kafirin,beta-kafirin are highly expressed in endosperm.DNA mutation analysis showed purification and selection effect.SNP and INDEL variations of kafirin genes have been identified for Chinese breeding lines Ji2731 and LR9198.3.In callus induction of mature seeds,different genotypes and different hormone ratios had significant effects on callus induction.The results showed that under the optimal medium MS+45 g/L sucrose +500 mg/L hydrolyzed casein+4.0 mg/L 2,4-D+0.2 mg/L KT+1.2% AGAR,the induction rate and differentiation rate were 82.86% and 0.The induction rate and differentiation rate of SSA were 60.71% and 17.78% respectively.The induction rate of 2083 was 38.17%,and the differentiation rate was 0.The induction rate and differentiation rate of R111 were 46.50% and 0.For the combination of 3 chi 3 and SSA with low concentration,the use effect of 2,4-D and KT is better than that of 2,4-D with low concentration,and also better than that of 2,4-D and KT with high concentration.For the combination of 2,4-D and KT with low concentration of 2083,the effect is better than the single use of 2,4-D with low concentration,but not as good as the combination of 2,4-D and KT with high concentration.The combination of 2,4-D and KT with low concentration of R111 is not as good as the single use of 2,4-D with low concentration,nor as good as the combination of 2,4-D and KT with high concentration.4.Genotype has a significant effect on callus induction in immature embryo.The results showed that the induction rate and differentiation rate were 74.15% and 0 % respectively.The induction rate and differentiation rate of SSA embryo were 78.81% and 27.96% respectively.The induction rate and differentiation rate of 2083 young embryo were 54.69% and 0.The induction rate and differentiation rate of R111 embryo were 96.51% and 0.5.In the process of sorghum tissue culture,different explants had different callus induction and differentiation rates.The results showed that the ability of callus induction and differentiation of immature embryos was stronger than that of mature seeds.Therefore,young embryo is an ideal material for the establishment of regeneration system.6.Kafirin promoter-gus A construct was used to co-culture young sorghum seeds with Agrobacterium tumefaciens.Expression of introduced genes in endosperm has been demonstrated by GUS staining. |
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