Inhibitory Effects of Plant Trypsin Inhibitors Msti-94 and Msti-16 on Therioaphis trifolii (Monell) (Homoptera: Aphididae) in Alfalfa.

Hailong Zhao,Hidayat Ullah,Mark Richard Mcneill,Kun Hao,Zehua Zhang,Guilin Du,Xiongbing Tu

doi:10.3390/insects10060154

Abstract

The spotted alfalfa aphid (Therioaphis trifolii (Monell)) is a known destructive pest that can significantly reduce alfalfa yields. Two differentially up-regulated alfalfa trypsin inhibitors ‘Msti-94’ and ‘Msti-16’ in transcriptome were verified in terms of their mRNA levels using RT-qPCR. The prokaryotic expression vector was constructed and its biological functions, including phenotypic and physiological responses, were verified through feeding spotted alfalfa aphids with active recombinant protein mixed with an artificial diet. Gene clone and gene prokaryotic expression confirmed that Msti-94 had a size of 651 bp, encoded 216 amino acids with a predicted protein weight of 23.5 kDa, and a pI value of 6.91. Similarly, the size of Msti-16 was 612 bp, encoded 203 amino acids, and had a predicted protein weight of 22.2 kDa with a pI value of 9.06. We concluded that both Msti-94 and Msti-16 acted as a stomach poison with survival rates reduced to 21.7% and 18.3%, respectively, as compared to the control, where the survival rate was significantly (p < 0.05) higher (60.0%). Aphid reproduction rates were significantly reduced, after 72 h of feeding, in both the Msti-94 and Msti-16 treatments compared to the controls. A concentration of 800 μg/mL (0.8 mg/mL) of recombinant protein and 5000 μg/mL (5 mg/mL) of recombinant expressing bacteria that inhibits the total protease, which ultimately disrupted the activity of trypsin, chymotrypsin, and aminopeptidase.

Highlights

Therioaphis trifolii (Monel) (Homoptera: Aphididae), the spotted alfalfa/clover aphid, often gathers on the lower parts of leaves and tender buds, where its prickly mouthpiece pierces the plant tissue and feeds on plant juice, causing the discoloration and yellowing of leaves
ribonucleic acids (RNAs) was was extracted extracted from the freshly picked leaves of alfalfa, alfalfa, which which clearly clearly showed of showed the the 5S, 5S, 18S, 18S, and and 28S
Plant protease inhibitors are a class of peptides or proteins that are known for their proteolysis

Summary

Introduction

Therioaphis trifolii (Monel) (Homoptera: Aphididae), the spotted alfalfa/clover aphid, often gathers on the lower parts of leaves and tender buds, where its prickly mouthpiece pierces the plant tissue and feeds on plant juice, causing the discoloration and yellowing of leaves. This restricts plant growth, causing senescence, referred to as the “green vein syndrome” [1,2]. The spotted alfalfa aphid was first reported in New Mexico, United States of America, where it had caused the wide-scale destruction of alfalfa crops [4]. The aphid is found in the regions of Hebei, Shandong, Henan, Shanxi, Gansu, Yunnan, Jilin, Liaoning, the Beijing municipality, and Mongolia, where infestations have been shown to cause an overall economic value reduction in more than 60% of alfalfa crops [5].

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