Bacillus Subtilis QM3 Induction On Alpha-amylase During Wheat Seed Germination

The rapid germination of seeds directly affects the yield and quality of wheat.At present,the use of microorganisms to promote seed germination has become a research hotspot.B.subtilis QM3 is a plant rhizosphere growth-promoting bacterium with independent intellectual property rights.The preliminary experimental results show that it has the functions of prevention,growth promotion and colonization.This paper mainly focused on the change of morphological indicators,α-amylase activity and isoenzymes during wheat seed germination after treatment with B.subtilis QM3,the relationship between B.subtilis QM3inducedα-amylase activity and GA and ABA.Study on the colonization of B.subtilis QM3during wheat germination,the production ofα-amylase by B.subtilis QM3 and its enzymatic properties.as follows:（1）EDTA（α-amylase inhibitor）and Ca Cl₂（α-amylase enhancer）were used to study the relationship between B.subtilis QM3 and wheat seed germination.The results showed that EDTA inhibited the germination of wheat seeds;Both B.subtilis QM3 and Ca Cl₂can promote the germination of wheat seeds,and the promotion is more obvious when the concentration of B.subtilis QM3 is 10⁶CFU/m L.After soaking the seeds with EDTA,compared with the germination group with sterile water,the germination rate and germination potential of the 10⁶CFU/m L B.subtilis QM3 germination group were significantly improved（p<0.05）,which indicates that B.subtilis QM3 can alleviate the inhibition of EDTA on wheat germination.The germination rate and germination vigor of the Ca Cl₂and B.subtilis QM3co-treatment group increased more significantly than the Ca Cl₂or B.subtilis QM3 treatment alone.The effects of different treatments on the bud length,root length,fresh weight,dry weight,and root-to-shoot ratio in wheat germination were basically the same as those on the germination rate and germination vigor.It shows that B.subtilis QM3 and Ca Cl₂have the same effect and can promote the germination of wheat seeds.（2）EDTA（α-amylase inhibitor）and Ca Cl₂（α-amylase enhancer）were used to study the relationship between B.subtilis QM3 andα-amylase in wheat seed germination（0-72 h）,and the DNS method was used to determineα-amylase active.The results showed that with the increase of germination time,theα-amylase activity in wheat seeds continued to increase,and the activity was the highest at 72 h;Compared with the control group,both B.subtilis QM3 and Ca Cl₂could increaseα-amylase activity.B.subtilis QM3 was more obvious when the concentration was 10⁶CFU/m L,andα-amylase activity increased by 16.88±0.28%at 72 h.After soaking seeds with EDTA,compared with the germination group with sterile water,theα-amylase activity of wheat seeds in the 10⁶CFU/m L B.subtilis QM3 germination group increased by 19.84±0.32%at 72 h,which alleviated the effect of EDTA pretreatment on the Inhibition ofα-amylase activity;Ca Cl₂and B.subtilis QM3 cross-treatment group wheat seedα-amylase activity is the highest.It shows that B.subtilis QM3 can induce the production ofα-amylase during wheat seed germination.（3）B.subtilis QM3 can induceα-amylase during wheat seed germination.Therefore,non-denaturing polyacrylamide gel electrophoresis was used to study the effect of10⁶CFU/m L B.subtilis QM3 onα-amylase isoenzymes during wheat seed germination.The results showed that there are threeα-amylase isoenzymes in wheat seeds,namely Aα,Bαand Cα.B.subtilis QM3 can increase the activity and content ofα-amylase isoenzymes during wheat seed germination,especially the Cαband ofα-amylase isoenzymes.（4）In wheat seed germination,α-amylase is regulated by hormones,so 10⁶CFU/m L B.subtilis QM3,20 mg/L GA₃,10 mg/L ABA,30 mg/L S-3307 were used to treat the germination process of wheat seeds to study the relationship betweenα-amylase induced by B.subtilis QM3 and hormones.α-amylase activity was determined by the potassium iodide plate method and DNS.The results show that both B.subtilis QM3 and GA₃can induce the production ofα-amylase.S-3307,an inhibitor of GA₃,can inhibit the induction ofα-amylase by GA₃,but cannot inhibit the induction ofα-amylase by B.subtilis QM3.It is speculated that the induction of wheat seedα-amylase by B.subtilis QM3 may not be regulated by changing the GA content in wheat seeds.ABA can inhibit the induction ofα-amylase by GA₃,and ABA cannot inhibit the induction ofα-amylase by B.subtilis QM3.It is speculated that the induction of wheat seedα-amylase by B.subtilis QM3 may not be affected by ABA.（5）DNS method and rifampicin resistance labeling method were used to study the α-amylase production of B.subtilis QM3 and its colonization in wheat germination.The results showed that:B.subtilis QM3 can colonize wheat during germination,and B.subtilis QM3 has the ability to produceα-amylase.The activity of the producedα-amylase is 50.58 IU/m L.The optimal reaction temperature of theα-amylase produced is 70°C,and its stability is high at 30-70°C.The optimal reaction p H is 6.0,and its stability is high at p H6.0-7.0.Ca²⁺,Na⁺,K⁺and Mg²⁺can promote the activity ofα-amylase,and Fe²⁺,Zn²⁺,Cu²⁺,Mn²⁺and EDTA can inhibit the activity ofα-amylase.From the above,B.subtilis QM3 can induceα-amylase in wheat seed germination and increaseα-amylase isoenzymes to promote wheat seed germination;It is speculated that the induction ofα-amylase in wheat germination by B.subtilis QM3 may not be regulated by changing the content of GA or may not be affected by ABA;B.subtilis QM3 can colonize wheat,and B.subtilis QM3 can produceα-amylase,thereby inducingα-amylase in wheat seed germination to promote wheat seed germination.