Study On Extraction,Purification Of Mung Bean Trypsin Inhibitor And Mechanism Of Resistance To Callosobruchus Chinensis(L.)

Trypsin inhibitor(TI)is a type of peptide or protein that inhibits trypsin.It has a high content in legumes seeds and it not only has anti-insect effects,but also has anti-virus and anti-tumor effects.Therefore,it is of great theoretical value and application value to search for new and highly active trypsin inhibitors.At present,the research on trypsin inhibitors is mainly concentrated in a few kinds of beans such as soybeans and cowpeas.And there are no reports on the activity level and insecticide mechanism of insect-resistant mung bean trypsin inhibitor(MBTI).For this reason,different mung beans are used as experimental materials in this study.The extraction,purification and stability of insect-resistant mung bean trypsin inhibitors were studied,and the highly active trypsin inhibitors were selected,and insect-resistant mung bean trypsin inhibitors were used to study the protease,detoxification enzymes,protective enzymes,and growth and development to the larvae of Callosobruchus chinensis.The aim of this study was to clarify the stability of mung bean trypsin inhibitor and its insecticidal mechanism to Callosobruchus chinensis,in order to provide theoretical basis for the exploitation and utilization of insect-resistant mung bean resources.The findings are as follows:1.The stability test of mung bean trypsin inhibitors showed that different temperatures,different pH,different amplitude ultrasound,different high temperature and high pressure,denaturant and reducing agent had certain effects on the residual activity of trypsin inhibitors in 5 mung bean varieties.And the longer the processing time,the greater the impact.Among them,insect-resistant mung bean B18 and B20 is is one of the two varieties with high stability of trypsin inhibitor,which has great application value2.The isolation and purification tests of mung bean trypsin inhibitors show that the optimum extractant for rough extraction of mung bean trypsin inhibitors is sodium chloride solution,and the optimum extraction time is 12h,Among them,the protein content,total activity and specific activity of insect-resistant mung bean B18 trypsin inhibitor crude extract were the highest.96.252 MG,553.738 U and 5.753 U/mg,respectively.When the optimal ammonium sulfate saturation was 60%,The obtained crude protein was isolated and purified by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 gel filtration chromatography to isolate trypsin inhibitors.The results showed that the trypsin inhibitor activity of insect-resistant mung bean was significantly higher than that of susceptible varieties.Among them,the trypsin inhibitor of B18 has the highest activity and purification multiple.with 88.766 U/mg and 14.641 times,respectively,B20 followed.At the same time,the molecular weight of trypsin inhibitors in 5 mung bean varieties was determined to be approximately 8.8 KDa.3.Effect of protease inhibitors on protease activity in Callosobruchus chinensis larvae show that,protease inhibitors(MBTI,PMSF,TLCK and OI)had certain inhibitory effects on the total protease,trypsin-like enzyme,and Chymotrypsin-like enzyme activities of Callosobruchus chinensis larvae in vitro Among them,MBTI with 20 ug/mL had the strongest inhibition effect on 3 enzyme activities;After Callosobruchus chinensis larvae ate artificial mung beans containing different inhibitors,the activities of three enzymes were also inhibited to some extent.With the prolongation of the age,the three enzyme activities increased but were still significantly lower than the control,and MBTI was the most inhibitory.4.The determination of enzyme activity in the larvae of Callosobruchus chinensis at different instars showed after Callosobruchus chinensis larvae feeding artificial mung beans containing 2.0%contents of MBTI,the inhibition of activities of the total protease,trypsin-like enzyme,and Chymotrypsin-like enzyme in larvae of different ages was the strongest.the induction of CarE and GSTs activity in larvae was the strongest.The inhibitory effect on SOD and CAT activity of Callosobruchus chinensis larvae at different ages was the strongest,and the induction of POD activity in larvae was the strongest.5.Continuous growth and development of Callosobruchus chinensis and determination of detoxification enzymes and protective enzyme activities in larvae showed that after four consecutive generations of Callosobruchus chinensis larvae feeding artificial mung beans containing MBTI,the larval weight and adult feathering rate were significantly reduced,and the development period was significantly prolonged.The activity of CarE,GSTs and POD in larvae was significantly induced and the activity of SOD and CAT in larvae was inhibited significantly.By the fourth generation,MBTI had the highest influence on its larval weight,adult feather rate,and developmental period;The induction of CarE,GSTs and POD activity in larva was the strongest in the second generation,the fourth generation and the third generation,respectively.The inhibition of SOD and CAT activity was the strongest in the first and third generation,respectively.The activity of insect-resistant mung bean trypsin inhibitor were significantly higher than that of susceptible variety,among which B18,B20 was the two varieties with high activity of mung bean trypsin inhibitor,which was of great application value.Mung bean trypsin inhibitor can affect the normal growth and development of Callosobruchus chinensis larvae by inhibiting activity of protease,detoxification enzymes and protective enzyme of Callosobruchus chinensis larvae,this will provide a theoretical basis for the study of the insect-resistant mechanism of mung bean trypsin inhibitors and scientific basis for the exploitation and utilization of insect-resistant mung bean resources.