Search

In Bacillus thuringenesis(Bt) transgenic cotton, the cotton boll has the lowest insecticidal protein content when compared to the other organs. The present study investigated the effects of amino acid spray application at the peak flowering stage on the cotton boll Bt toxin concentration and yield formation. Boll protein synthesis and carbohydrate conversion were also studied to reveal the fundamental mechanism. Three treatments (i.e., CK, the untreated control; LA1, five amino acids; LA2, 21 amino acids) were applied to two Bt cultivars of G. hirsutum(i.e., the hybrid Sikang 3 and the conventional Sikang 1) in the cotton-growing seasons during 2017 and 2018. Amino acid spray application at the peak flowering stage resulted in an increase of 5.2–16.4% in the boll Bt protein concentration and an increase of 5.5–11.3% in the seed cotton yield, but there was no difference between the two amino acid treatments. In addition, amino acid applications led to increases in the amino acid content, soluble protein content, glutamate pyruvate transaminase (GPT) activity, glutamate oxaloacetate transaminase (GOT) activity, glucose content, fructose content and soluble acid invertase (SAI) activity. This study also found that Bt protein content, enhanced boll number and the weight of opened bolls were closely related to carbon and nitrogen metabolism. The Bt protein content had significant linear positive correlations with amino acid and soluble protein contents. Enhanced boll number had significant linear positive correlations with the GPT and GOT activities from 15–25 days after flowering (DAF). The weight of opened bolls from 55–65 DAF had a significant linear positive correlation with the SAI activity. These results indicate that the enhancement of boll protein synthesis and carbohydrate conversion by amino acid application resulted in a simultaneous increase in the boll Bt protein concentration and cotton lint yield.

Bt cotton production is mainly challenged by two problems, i.e., the low concentration of Bacillus thuringiensis(Bt) protein at boll setting stage and its lowest insect resistance in bolls of all organs. Therefore, increasing Bt protein concentration at boll stage, especially in bolls, has become the main goal in increasing insect resistance in cotton. In this study, two protein degradation inhibitors (EDTA and Leupeptin) were sprayed on the bolls, subtending leaves, and whole cotton plants respectively at peak flowering stage of two Bt cultivars (medium maturation Sikang 1 (SK1) and early maturation Zhongmian 425 (Z425)) in 2019 and 2020, respectively. The Bt protein content and protein degradation metabolism were investigated. The results showed that the Bt protein concentration enhanced by 21.3 to 38.8%, 25.0 to 38.6% in treated bolls of SK1 and ZM425 respectively, while decreasing in the subtending leaves of these treated bolls. In the treated leaves, the Bt protein concentration increased by 7.6 to 23.5%, 11.2 to 14.9% for SK1 and Z425, respectively. The combined application of EDTA and Leupeptin to the whole cotton plants increased the Bt protein concentration in both bolls and subtending leaves. The Bt protein concentration in bolls was higher, increasing by 22.5 to 31.0%, 19.6 to 32.5% for SK1 and ZM425 respectively. The organs treated with EDTA or/and Leupeptin showed reduced free amino acid content, protease and peptidase activities and a significant enhancing in soluble protein content. These results indicated that inhibiting of protein degradation could improve protein content, thus increasing the Bt protein concentration in bolls or/and leaves of cotton plants. Therefore, the increase in the Bt protein concentration without yield reduction suggested that the two protein degradation inhibitors may applicable for insect resistance in cotton production.

The effects of mepiquat chloride (DPC) on Cry1Acprotein contents in Bacillus thuringiensis(Bt) cotton boll shells under high temperature and drought stress were investigated to provide a theoretical reference for Bt cotton breeding and high-yield and high-efficiency cotton cultivation. This study was undertaken on the Bt cotton cultivar ‘Sikang 3’ during the 2020 and 2021 growing seasons at Yangzhou University Farm, Yangzhou, China. The potted cotton plants were exposed to high temperature and drought stress, and 20 mg L−1DPC and water (CK) were sprayed on cotton plants. Seven days after treatment, Cry1Acprotein content, α-ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content were measured, and transcriptome sequencing was performed. DESeq was used for differential gene analysis. Under the DPC treatment, in contrast with the water treatment (CK), Cry1Acprotein content increased by 4.7-11.9%. α-Ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content all increased. Transcriptome analysis revealed that there were 7542 upregulated genes and 10449 downregulated genes for DPC vs. CK. GO and KEGG analyses showed that differentially expressed genes were mainly involved in biological processes, such as amino acid metabolism and carbon metabolism. Genes coding 6-phosphofructokinase, pyruvate kinase, glutamic pyruvate transaminase, pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, glutamate synthase, 1-pyrroline-5-carboxylate dehydrogenase, glutamic oxaloacetic transaminase, amino-acid N-acetyltransferase, and acetylornithine deacetylase were all significantly upregulated. DPC increased pyruvate, α-ketoglutarate, and oxaloacetate by increasing the operating rate of the glycolytic pathway, the citric acid cycle. It also significantly up-regulated the genes encoding glutamate synthase and pyrrolidine-5-carboxylic acid dehydrogenase, and down-regulated the genes encoding glutamine synthetase as well as up-regulated the genes encoding glutamate oxaloacetate transaminase and N-acetylglutamate synthetase. Therefore, the synthesis ability of aspartic acid, glutamic acid, pyruvate, and arginine was increased by DPC, and then Cry1Acprotein contents were increased by regulating carbon and amino acid metabolism.

To clarify the effect of the N deficit on the amount of square Bt insecticidal protein, different N application rates (0, 75, 150, 225, and 300 kg ha–1) were imposed on the conventional cultivar Sikang 1 (SK-1) and hybrid cultivar Sikang 3 (SK-3) during 2015–2016 cotton growth seasons. Under different N application rates, the square number per plant, square volume and square dry weight reduced when the N rates decreased from conventional rate (300 kg ha–1) to 0 kg ha–1. And the square Bt protein content decreased accordingly. The analysis of N metabolism showed that soluble protein content, GPT and GOT activities decreased, free amino acid, peptidase and protease activities increased under N deficit. Correlation analysis indicated that the reduced Bt protein content under N deficit was related to altered N metabolism. In conclusion, square development and the amount of square Bt toxin both decreased under N deficit, indicating that promoting the square development under appropriate N application rate would also promote the insect resistance during squaring stage.