Research On The Resistance Of The Insect-Specific Neurotoxin LqhIT2and LMX From Scorpion To The Rice Leaf Rollers

Rice (Oryza sativa) acts as a staple food for much of the global population, particularly in Asia where about90%of people live on rice. However, in commercial production, rice productivity and quality are adversely affected by many biotic stresses, particularly insect pests. Rice leaf folder (Cnaphalocrocis medinalis Guenee), which is one of the most common insece pest of rice yields, is an insect with migratory characteristics. In China, since2003, the annual average area damaged by rice leaf folder was more than20million hm2and the grain yield loss was up to760million kg every year.To avoid rice yield loss caused by leaf folder infestation, conventional means rely on the extensive use of chemical pesticides and Bacillus thuringiensis (Bt) gene application. However, chemical pest control is environmentally unfriendly and pest-resurgence often occurs. Meanwhile, long-term use of Bt genes will increase the risk of narrowing the insect-resistant spectrum of transgenic plants. Thus, to meet this challenge, there is an urgent need to explore economically and ecologically sound alternatives so as to enrich genetic diversity. Because these scorpion toxins are neuromuscular system-specific in insects and are safe for mammal, it has a significant potential for the development of safe insecticides with a broad spectrum.The anti-insect scorpion toxin LqhIT2, identified in Leiurus quinquestriatus hebraeus, is a long chain depressant insect toxin. The amino acid of mature LqhIT2polypeptide included61amino acid residues cross-linked by4disulphide bridges, which principally affect sodium channels in excitable cells. It is certified that LqhIT2has high and special effectivity against lepidoptera insects without apparent toxicity to mammals. However, there is no report about LqhIT2transgenic research. In our study, we introduced LqhIT2gene into indica rice Yuetai B to investigate the resistance to rice leaf folder. The results of transgenic rice resistance to pest showed that the damage rate of LqhIT2transgenic rice plants were decreased comparing to the wild type plants. Meanwhile, Furthermore, the analysis of plant hormone and metabolites in LqhIT2transgenic rice plants suggested that, compared with controls, ectopic expression of LqhIT2profoundly activated the expression level of LOX which is very important for jasmonic acid biosynthesis pathway and enhanced the content of linolenic acid, cis-(+)-12-oxophytodienoic acid, jasmonic acid and jasmonic acid isoleucine. Besides, in LqhIT2plants, the expression level of PAL which encode key enzyme involved in phenylpropanoid pathway was also up-regulated, and secondary metabolites of flavonoid and lignins which were downstream products of phenylpropanoid pathway were also increased in LqhIT2transgenic plants. Parellel to this is the down-regulated expression level of starch, glucose and glucose-6-phosphate, related to glycolysis pathway that supplys intermediate carbon products required for plant secondary metabolites, and genes encoding amylase, hexokinase (HK) and fructokinase (FK) which were key enzymes in glycolysis pathway were significantly up-regulated. Based on the data above, we concluded that expression of LqhIT2could up-regulate the content of linolenic acid and expression level of LOX to activate jasmonate biosynthese. Jasmonate primes of the expression level of PAL to active phenylpropanoid pathway and produce flavonoids and lignin to increase the rice resistance to rice leaf folder.LMX is the artificial optimization peptide based on LqhIT2, which shares more than85%amino acid and92%nucleotide homology with LqhIT2. In the study of LMX, compared with controls (PBS buffer and10(?g GST protein), oral ingestion of LMX protein (2.5?g,5.0?g,7.5p?g and1O?g) led to a significant decrease in feeding on rice leaves, an obvious repression of larval growth and development, delay in molting. On the lethality of larval, oral ingestion of LMX proteins cause an obvious increase in larval lethality and the lethality of LMX was higher than that of LqhIT2. Biochemical analysis showed that LMX protein ingestion dramatically decreased ecdysone content in the larvae of rice leaf folder, down-regulated enzymatic activities of the detoxification system (a-naphthyl acetate esterase and glutathione S-transferase), the digestive system (tryptase and chymotrypsin), and the antioxidant system (catalase) in rice leaf folder compared to the controls. Transgene analysis showed that, compared with the wild type, the average damage rate of the LMX transgenic rice leaf was decreased more than30%. Under artificial and natural infestation, the damaged tillers of transgenic lines was decreased14.7%-29.2%and30.6%-48.3%, and the damaged leaves of transgenic lines was decreased19.3%-39.1%and19.1%-36.9%relative to those of the wild type group, respectively. However, no significant differences were observed in the damage rates of tillers and leaves between wild type and rbcS plants. Above all, the stable LMX protein results in disrupted enzyme activities and delay in larva development, it is critical for improving resistance to rice leaf folder.