1. Nutrient Insecticidal Protein Genes It is well known that Bacillus thuringiensis is a gram-positive Agrobacterium that produces the insecticidal parasporal crystal protein called delta-endotoxin during sporulation. These proteins are highly The insecticidal activity. In the past few decades, dozens of Bacillus thuringiensis strains and more than 130 of the insecticidal crystal proteins they encode have been identified. In recent years, cloned Bt genes have been transferred to plants and expressed highly in plants. In spite of this, Bt insecticidal protein is not effective for certain agriculturally important pests, such as Lepidoptera. In the world, small ground tigers endanger 50 kinds of crops, and the losses caused by their larvae are often irretrievable. In research, it was found that during the vegetative growth stage before sporulation, another non-δ-endotoxin-like insecticidal nutrient protein, the vipative insecticidal protein (Vip), was secreted and produced. Generation of insecticidal protein. At present, three nutrient insecticidal proteins have been discovered: Vip1, Vip2 isolated from B. cereus cultures, and Vip3A isolated from cultures of B. thuringienses, VipN The terminal sequence contains a region of several positively charged amino acid residues and a hydrophobic core region. Vip proteins bind to sensitive insect epidermal cells, especially columnar cells, causing cell disintegration and severe damage to the intestine, causing insects to rapidly die. Symptoms caused by insects feeding on Vip3A are similar to those caused by δ-endotoxins, but the duration of Vip3A is longer. At present, the Vip3A gene has been cloned and its research has been deepened. Juan et al. (1996, 1997) isolated and cloned Vip3A(a) and Vip3A(b) genes from Bacillus thuringiensis strains AB88 and AB424 respectively, and the amino acid homology of their expressed products was as high as 98%. Vectors constructed from these two genes have been expressed in E. coli. The researchers fed young hatchling larvae with an E. coli ultrasonic extract containing the Vip3A(a) and Vip3A(b) genes. After 6 days, the larvae did not survive, whereas the control larvae survived the majority. The Vip3A gene is 2.4 kb in length and encodes a 791 amino acid (88.5 kDa) insecticidal protein with no homology to any known known protein. Studies also found that Vip3A(a) is a secreted protein that is present in the supernatant of AB88 cultures. The secretory protein crosses the wall of the bacterium and requires two consecutive steps, first the insertion of the precursor protein into the cell membrane, followed by the secretion of the protein across the cell wall to complete the transmembrane transport of the protein. Westem hybridization analysis showed that Vip3A(a) protein could be detected in cultured AB88 for 15 hours. The expression of Vip3A(a) protein was highest at the early stage of stable growth and maintained until the sporulation period. However, δ-endotoxin needs to be cultured for 36 hours before it can be detected. Therefore, the molecular and biological characteristics of the Vip3A(a) nutrient insecticidal protein are different from those of the Btδ-endotoxin family. Biological test results show that Vip3 has a broad-spectrum Lepidopteran insecticidal activity, especially for small ground tiger, armyworm and Spodoptera exigua. The effective concentration of Vip (30-100 ng) is similar to that of the Bt insecticidal protein. (Li Huifen Zhu Xi) 2, cholesterol oxidase gene cholesterol oxidase gene (cholesterol oxidase, Cho) is also known as the second generation of insect-resistant genes, its expression product is a new class of insecticides. Cholesterol oxidase is found in bacteria such as Streptomyces, Brevibacterium, Pseudomonas, and Schizopylium. The cholesterol oxidase gene has been isolated from the genus Streptomyces. The encoded cholesterol oxidase belongs to the acetylcholine oxidase family and catalyzes the formation of 17-ketosteroids and hydrogen peroxide. Cholesterol is the main component of the cell membrane. Along with the above reaction, the intestinal epithelial cells of the ingested insect exhibit cytolysis, thereby causing insect death. David et al. (1994) isolated the cholesterol oxidase gene (ChoM) from Streptomyces A19249. The gene encoding ChoM is 1512 bp in length and encodes 504 amino acids. The original ChoM encoding gene carries a 129 bp leader at the 5' end and encodes a 43 amino acid short peptide. The 5-terminally modified full-length ChoM gene without the leader sequence was constructed into the vector pKK233-2 to obtain two expression vectors and was found to be detectable in the E. coli culture fluid of the full-length ChoM gene with the leader sequence. High levels of cholesterol oxidase activity, while cholesterol oxidase activity was barely detectable in E. coli with only the ChoM encoding gene. The researchers constructed them on the plant expression vector pMON1772 and transformed the tobacco protoplasts with electroporation. It was found that the expression of the full-length cholesterol oxidase gene with the leader sequence was 2-5 times higher than that of the ChoM coding gene alone. This indicates that the N-terminal leader sequence may have a significant enhancement of protein expression. The results of biological tests showed that cholesterol oxidase had obvious toxicity to Boll weevil and Tobacco budworm, and its effective concentration was similar to that of Bt insecticidal crystal protein. Boll weevil is one of the major cotton pests in the world. The site of damage is cotton bolls. Adult eggs are produced in cotton buds. The larvae complete the entire development process on cotton bolls. Because conventional chemical agents are difficult to reach within the flower buds, it is difficult to control such pests, and the use of insecticidal proteins produced by the transgenic cotton itself can achieve an ideal insecticidal effect. At present, the commonly used Bt insecticidal protein gene cotton has obvious control effect on the cotton bollworm, but it has little effect on the boll weevil and cholesterol oxidase may make up for this deficiency. In short, the current plant insect-resistant genetic engineering is a hot topic of research. Humans are making unremitting efforts to find new insect-resistant genes. The new insecticidal genes will continue to be discovered. It is of great significance that this insect-resistant transgenic plant will be replaced with new generations to prevent pests from developing resistance to certain insect-resistant transgenic plants. The development trend is to transfer more than one type of insect-resistant gene at the same time to obtain a transgenic plant having a wide insect-resistance spectrum, strong insect resistance, and insects difficult to produce tolerance.
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