Screening Of Alfafa Trypsin Inhibitor And The Inhibiting Effect On Therioaphis Trifolii Monel(homptera: Aphididae)

Protease inhibitors are a class of small molecular peptides or proteins that widely exist in animals,plants and microbes,which can regulate the balance of proteolytic enzymes in the bodies,and combine protease allosteric site with active sites,inhibits the catalytic activity of proteolytic enzymes.The trypsin inhibitor gene of alfalfa was cloned in this paper,the recombinant expression vector was constructed,and the recombinant protein was purified.The effects of Therioaphis trifolii on the growth and development were studied.The results showed that the recombinant protein had a good inhibitory effect on Therioaphis trifolii.The enzyme activity in aphids were significantly inhibited.The conclusions of this study are as follows:Six differentially expressed alfalfa trypsin inhibitor genes were screened in the transcriptome sequencing experiment of alfalfa deal with the therioaphis trifolii.After real-time quantitative PCR validation,the sequence of the gene of ATG initiation site and TAA termination site were analyzed according to the results of NCBI comparison.Two oligonucleotide primers were designed and synthesized for two alfalfa trypsin inhibitor genes.The total genomic c DNA of alfalfa leaves was used as template.Two Kunnitz type trypsin inhibitor genes were cloned by polymerase chain reaction(PCR).The length of the two genes was about 657 bp and 612 bp.The amplified target fragment sequence is inserted into p MD19-T,transform into BL21(DE3)after screening positive clones transformants.It's proved that the sequence was a full-length sequence of the desired target gene and named as Msti-95(Medicago sativa trypsin inhibitor 95),Msti-16(Medicago sativa trypsin inhibitor 16).Two oligonucleotide primers containing restriction endonuclease Bam HI and Xho I sites were designed and synthesized.The target fragments were amplified by PCR.The recombinant plasmids p T-Msti-95 and p T-Msti-16 were obtained by antibiotic screening after transformation.The target fragment and expression vector p SYno-1 were digested with restriction endonuclease Bam HI and Xho I,and positive colony plasmids were extracted by T4 DNA-ligase.Msti-95 and Msti-16 genes have been identified by restriction endonuclease digestion and sequencing analysis.The recombinant expression vectors p S-Msti-95 and p S-Msti-16 were constructed.The expression of the target gene was induced chemically by IPTG,and the optimal expression conditions were screened.The electrophoretic analysis of the total protein of BL21(DE3)containing the expression vector showed that the foreign gene was highly expressed.The purified protein was purified by Ni column.The purified protein was eluted by Wash Buffer containing imidazole.The protein was detected by protein electrophoresis and the expressed protein was obtained with high purity.The effects of protein inhibitor Msti-95 and Msti-16 on the survival,growth and development of alfalfa aphid were determined by feeding method.The results show that It has good gastric toxicity to alfalfa aphid.The survival rates of alfalfa aphid on the artificial diet with Msti-95 and Msti-16 protein(800 ?g/ml)were 21.7%(P=0.04)and18.3%(P=0.03),respectively,which were significantly lower than the survival rates of control group 60.0%.The reproductive rate was 186.7%(P=0.04)and 198.3%(P=0.05),both of which were significantly lower than the reproductive rate of the control group 315.0%.The effect of protease activity on the activity of protease in the insect was determined by Elisa.The results showed that the activity of total protease,trypsin chymotrypsin and aminopeptidase in alfalfa aphid were significantly lower than the control group(P<0.05)after alfalfa aphid was fed with purified protein(800 ?g/m L)mixed with pure artificial feed.As same as the purified protein,the activity of total protease,trypsin chymotrypsin and aminopeptidase in alfalfa aphid were significantly lower than the control group(P<0.05)after alfalfa aphid was fed with the bacteria expressed protein(5000 ?g/m L)were significantly decreased(P<0.05).