Inhibition of protein degradation increases the Bt protein concentration in Bt cotton

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

Bt Cotton in China:Are Secondary Insect Infestations Offsetting the Benefits in Farmer Fields?

The area sown to Bt cotton has expanded rapidly in China since 1997. It has effectively controlled the bollworm. However, in recent years, concern has surfaced about the emergence of secondary insect pests, particular mirids, in Bt cotton fields. This study measures the patterns of insecticide use based on farm-level from 1999 to 2006, the analysis demonstrates a rise in insecticide use to control mirids between 2001 and 2004, secondary insect infestations is largely related to the rise of mirids, but this rising did not continue in more than half of sample villages studied in 2004-2006. Moreover, the increase in insecticide use for the control of secondary insects is far smaller than the reduction in total insecticide use due to Bt cotton adoption. Further econometric analyses show that rise and fall of mirids is largely related to local temperature and rainfall.

Effects of Extreme Air Temperature and Humidity on the Insecticidal Expression Level of Bt Cotton

The higher survival rates of Helicoverpa amigera larvae were usually observed after adverse climate which was related to extreme temperature （T） and relative humidity （RH） stresses in transgenic Bacillus thuringiensis （Bt） cotton. The unstable resistance of Bt cotton to bollworms has been correlated with the reduced expression of CrylAc δ-endotoxin. The objective of this study was to investigate the effects of combined temperature and relative humidity stresses on the leaf CrylAc insecticidal protein expression during critical developmental stages. The study was undertaken on two transgenic cotton cultivars that share same parental background, Sikang 1 （a conventional cultivar） and Sikang 3 （a hybrid cultivar）, during the 2007 and 2008 growing seasons at the Yangzhou University Farm, Yangzhou, China. The study was arranged with two factors that consisted of temperature （two levels） and relative humidity （three levels）. The six T/RH treatments were 37℃/95%, 37℃/70%, 37℃/50%, 18℃/95%, 18℃/70%, and 18℃/50%. In 2007, the six treatments were imposed to the plants at peak flowering stage for 24 h; in 2008, the six treatments were applied to the plants at peak square, peak flowering, and peak boll stages for 48 h. The results of the study indicated that the leaf insecticidal protein expression in CrylAc was significantly affected by extreme temperature only at peak flowering stage, and by both extreme temperature and relative humidity during boll filling stage. The greatest reductions were observed when the stresses were applied at peak boll stage. In 2008, after 48 h stress treatment, the leaf Bt endotoxin expression reduced by 25.9-36.7 and 23.6-40.5% at peak boll stage, but only by 14.9-26.5 and 12.8-24.0% at peak flowering stage for Sikang 1 and Sikang 3, respectively. The greatest reduction was found under the low temperature combined with low relative humidity condition for both years. It is believed that the temperature and relative humidity stresses may be attributed to the reduced efficacy of Bt cotton in growing conditions in China, where extreme temperatures often increase up to 35-40℃ and/or decrease down to 15-20℃, and relative humidity may reach to 85-95% and/or reduce to 40-55% during the cotton growing season.

Effects of soil salinity on rhizosphere soil microbes in transgenic Bt cotton fields

With increased cultivation of transgenic Bacillus thuringiensis （Bt） cotton in the saline alkaline soil of China, assessments of transgenic crop biosafety have focused on the effects of soil salinity on rhizosphere microbes and Bt protein residues. In 2013 and 2014, investigations were conducted on the rhizosphere microbial biomass, soil enzyme activities and Bt protein contents of the soil under transgenic Bt cotton （variety GK19） and its parental non-transgenic cotton （Simian 3） cultivated at various salinity levels （1.15, 6.00 and 11.46 dS m-1）. Under soil salinity stress, trace amounts of Bt proteins were ob- served in the Bt cotton GK19 rhizosphere soil, although the protein content increased with cotton growth and increased soil salinity levels. The populations of slight halophilic bacteria, phosphate solubilizing bacteria, ammonifying bacteria, nitrifying bacteria and denitrifying bacteria decreased with increased soil salinity in the Bt and non-Bt cotton rhizosphere soil, and the microbial biomass carbon, microbial respiration and soil catalase, urease and alkaline phosphatase activity also decreased. Correlation analyses showed that the increased Bt protein content in the Bt cotton rhizosphere soil may have been caused by the slower decomposition of soil microorganisms, which suggests that salinity was the main factor influencing the relevant activities of the soil microorganisms and indicates that Bt proteins had no clear adverse effects on the soil microorganisms. The results of this study may provide a theoretical basis for risk assessments of genetically modified cotton in saline alkaline soil.

Relationship Between Leaf C/N Ratio and Insecticidal Protein Expression in Bt Cotton as Affected by High Temperature and N Rate

Expression of insecticidal protein for transgenic Bacillus thuringiensis （Bt） cotton is unstable and related to nitrogen metabolism. The objective of this study was to investigate the relationship between leaf carbon nitrogen ratio （C/N） and insecticidal efficacy of two Bt cotton cultivars. C/N ratio and Bt protein content were both measured at peak square period and peak boll period respectively under 5-7 d high temperature and different nitrogen fertilizer rates on the Yangzhou University Farm and the Ludong Cotton Farm, China. All plants were grown in field. The results showed that the C/N ratio enhanced slightly and the Bt protein content remained stable at peak square period, but significant increases for the C/N ratio and decreases markedly for the leaf Bt protein concentration were detected at the peak boll period. The similar patterns at the two growth periods were found for the leaf C/N ratio and Bt protein content by different N fertilizer treatments. When nitrogen rate was from 0 to 600 kg ha-l, the C/N ratio was reduced by 0.017 and 0.006 for Sikang 1 and Sikang 3 at peak square period, compared to the 1.350 to 1.143 reduction for Sikang 1 and Sikang 3 at peak boll period, respectively. Correspondingly, the leaf Bt protein contents were bolstered by 2.6-11.8 and 26.9-36.9% at the two different growth periods, respectively. The results suggested that enhanced C/N ratio by high temperature and nitrogen application may result in the reduction of inseetiocidal efficacy in Bt cotton, especially in peak boll period.

The effects of high temperature level on square Bt protein concen-tration of Bt cotton

Higher boll worm survival rates were detected after high temperature presented during square period in Bt cotton. The objective of this study was to investigate the effects of high temperature level on the Bt efficacy of two different types of Bt cotton cultivars at squaring stage. During the 2011 to 2013 cotton growth seasons, high temperature treatments ranged from 34 to 44°C in climate chambers, and field experiments under high temperature weather with various temperature levels were conducted to investigate the effects of the high temperature level on square Bt protein concentration and nitrogen metabolism. The climate chamber experiments showed that the square insecticidal protein contents reduced after 24 h elevated temperature treatments for both cultivars, whereas significant declines of the square insecticidal protein contents were detected at temperature 〉38°C, and only slightly numerical reductions were observed when temperature below 38°C. Similar high temperature responses were also observed at the two field experimental sites in 2013. Correspondingly, high temperature below 38°C seems have little effect on the square amino acid concentrations, soluble protein contents, glutamic-pyruvic transaminase（GPT） and glutamic-oxalacetic transaminase（GOT） activities as well as protease and peptidase activities; however, when the temperature was above 38°C, reduced soluble protein contents, enhanced amino acid concentrations, decreased GPT and GOT activities, bolstered protease and peptidase activities in square were detected. In general, the higher the temperature is（〉38°C）, the larger the changes for the above compound contents and key enzymes activities of the square protein cycle. The findings indicated that the unstable insect resistance of the square was related to high temperature level during square stage.

Nitrogen spraying affects seed Bt toxin concentration and yield in Bt cotton

Cotton bolls exhibit the lowest insecticidal efficacy among all organs of Bt cotton,which would ultimately affect the yield formation.The objective of this study was to investigate the effects of different urea concentrations on the seed Bt protein contents,seed cotton yield and the corresponding protein metabolism mechanism.The experiments were conducted during 2017–2018 cotton growing seasons.Two cultivars,Sikang 3(hybrid,SK3)and Sikang 1(conventional,SK1),were treated with six urea concentrations and their seed Bt protein contents were compared during boll formation period.The urea spray concentration had a significant effect on the seed Bt toxin content and seed cotton yield.Spraying of either 5 or 6%urea led to higher insecticidal protein contents and higher seed cotton yield for both cultivars.Moreover,the highest amino acid and soluble protein contents,as well as GPT and GOT activities,and lower protease and peptidase activities were observed at the 5 to 6%urea levels.Significant positive correlations between the seed Bt toxin and amino acid contents,and between the seed Bt toxin content and GPT activities were detected.The lower boll worm number and hazard boll rate were also observed with the 5 to 6%urea treatments,which may be the reason why nitrogen spraying increased the seed cotton yield.Therefore,our results suggested that the seed Bt toxin content and insect resistance were impacted markedly by external nitrogen application,and 5 to 6%urea had the greatest effect on insect resistance.

Dynamics of Bt cotton Cry1Ac protein content under an alternating high temperature regime and effects on nitrogen metabolism

This study was conducted to investigate the effects of alternating high temperature on CrylAc protein content on Bt cotton cultivars Sikang 1 （SK-1, a conventional cultivar） and Sikang 3 （SK-3, a hybrid cultivar）. In 2011 and 2012, cotton plants were subjected to high temperature treatments ranging from 32 to 40℃ in climate chambers to investigate the effects of high temperature on boll shell insecticidal protein expression. The experiments showed that significant decline of the boll shell insecticidal protein was detected at temperatures higher than 38℃ after 24 h. Based on the results, the cotton plants were treated with the threshold temperature of 38℃ from 6：00 a.m. to 6：00 p.m. followed by a normal temperature of 27℃ during the remaining night hours （DH/NN） in 2012 and 2013. These treatments were conducted at peak boll growth stage for both cultivars in study periods of 0, 4, 7, and 10 d. Temperature treatment of 32℃ from 6：00 a.m. to 6：00 p.m. and 27℃ in the remaining hours was set as control. The results showed that, compared with the control, after the DH/NN stress treatment applied for 7 d, the boll shell CrylAc protein content level was significantly decreased by 19.1 and 17.5% for SK-1 and by 15.3 and 13.7% for SK-3 in 2012 and 2013, respectively. Further analysis of nitrogen metabolic physiology under DH/NN showed that the soluble protein content and the glutamic pyruvic transaminase （GPT） activities decreased slightly after 4 d, and then decreased sharply after 7 d. The free amino acid content and the protease content increased sharply after 7 d. The changes in SK-1 were greater than those in SK-3. These results suggest that under DH/NN stress, boll shell CrylAc protein content decline was delayed. Reduced protein synthesis and increased protein degradation in the boll shell decreased protein content, including Bt protein, which may reduce resistance to the cotton bollworm.

The impact of Bt cotton adoption on the stability of pesticide use

Even though the impact of Bacillus thuringiensis （Bt） cotton on pesticide use has been well documented, all previous stud- ies focus on the mean value of pesticide use. Using seven unique waves of panel data collected between 1999 and 2012 in China, we show that Bt cotton adoption has not only caused a reduction of the mean value of pasticide use, but also a reduction of the standard deviation of pesticide use. We conclude that Bt technology adoption has also contributed to the stability of pesticide use in cotton production. We believe that this contribution is theoretically and practically relevant because of the long length of our unique dataset.

Yield, Leaf Senescence, and Cry1Ac Expression in Response to Removal of Early Fruiting Branches in Transgenic Bt Cotton

Two-year field experiments were conducted at Linqing, Yellow River valley of China, to study the plant response to the removal of early fruiting branches in transgenic Bt （Bacillus thuringiensis） cotton （Gossypium hirsutum L.） from 2003 to 2004. Plants were undamaged and treated by removing two basal fruiting branches （FB） at squaring to form the control and the removal treatment, respectively. The plant height, leaf area （LA）, dry weight of fruiting forms （DWFF）, the number of fruiting nodes （NFN）, photosynthetic （Pn） rate, and levels of leaf chlorophyll （Chl）, N, P, K, and Cry lAc protein in main- stem leaves were measured at a 10- or 20-d interval after FB removal, and the sink/source ratio as indicated by NFN/LA and DWFF/LA was determined. FB removal significantly increased the plant height, LA, and plant biomass in both years. Lint yields were increased 7.5 and 5.2% by removal compared with their controls in 2003 and 2004, respectively. Significant increases in boll size （5.7 and 5.1%） were also observed in removal than in control for both years. Either NFN/LA or DWFF/LA was significantly reduced by removal before 40 d after removal; however, both NFN/LA and DWFF/LA were significantly enhanced by FB removal at 80 d after removal compared to the untreated control. There was no significant difference in fiber quality in the first two harvests between removal and control, but fiber strength and micronarie in the third harvest were significantly improved by FB removal. In terms of leaf Chl, Pn rate, levels of total N, P, and K in late season, leaf senescence was considerably delayed by FB removal. Levels of CrylAc protein in the fully expanded young leaves were considerably higher in FB-excised plants than in control, indicating FB removal enhanced CrylAc expression. It is suggested that the yield and quality improvement with FB removal may be attributed to the increased NFN/LA or DWFF/LA in late season and delayed leaf senescence, respectively. FB removal can be a potential practice incorporated into the intensive cultivation system for enhancing transgenic Bt cotton production.

Efficiency of cotton bollworm (Helicoverpa armigera Hübner) control of different Bt cotton varieties in North China

Background： The cotton bollworm （Helicoverpa armigera） is one of cotton＇s most destructive insect pests in terms of yield and quality. Since 1997, China has grown commercially available transgenic Bacillus thuringiensis （Bt） cotton. We aimed to investigate the variation in resistance of transgenic Bt cotton varieties to cotton bollworm in North China. Methods： Populations of cotton bollworm were monitored from 2008 to 2015 in environments where Bt cotton was planted adjacent to other non Bt crops. The study included 197 Bt cotton varieties planted in 42 counties/locations in three provinces （Hebei, Shandong and Henan） of North China, which were evaluated through field investigations, bioassays, and enzyme linked immunosorbent assays （ELISA）. Results： The average number of cotton bollworms never exceeded the action threshold （10 larvae per 100 cotton plants）, however, their number reached 19.55 per 100 cotton plants in 2011. The ratios of damaged plants to total Bt cotton stem tips, buds, and bolls was low except in 2010, when the ratios reached 1.82%, 2.09%, and 10.63%, respectively. The results of bioassay showed that the corrected mortality were higher at the second generation cotton bollworm stage than the third and fourth germination stages. Totally, Bt protein content declined sharply at the seedling stage from 2008 to 2015. Conclusions： This study indicated that almost all Bt cotton varieties were capable to effectively control the populations of cotton bollworm in North China.

Differential Expression of Bt Protein in Transgenic Bt Cotton

[Objective] The paper was to study the temporal and spatial dynamics of Bt protein expression in transgenic Bt cotton and to determine the inner relationship of Bt protein expression and transgenic Bt cotton. [Method] With transgenic cotton cultivar（ GK19） as the test material,Bt protein contents in different organs,main stem functional leaves at different growth stages and different positions of main stem leaves at different growth stages were studied by enzyme-linked immunosorbent assay. [Result] There were differences in Bt protein content among different organs of transgenic Bt cotton; the Bt protein content of leaves at seedling stage was the highest,followed by flowers,bubs and bolls,and those of roots and stems were relatively low. The Bt protein content of main stem function leaves gradually decreased with the progressing development. There were great differences in Bt protein content among different positions of main stem leaves at different growth stages; the Bt protein content of the 1^（st）-7^（th） top leaves at seedling stage and full budding stage gradually decreased,while those at full flowering stage and full bolling stage first slowly increased then gradually stabilized. [Conclusion] Bt protein expression was found in all organs of transgenic cotton at all growth stages,and the expression level presented temporal and spatial dynamics.

Spatial distribution of bolls affects yield formation in different genotypes of Bt cotton varieties

To optimize the spatial distribution of cotton bolls and to increase the yield,the relationship between yield components and boll spatial distribution was investigated among different Bt(Bacillus thuringensis)cotton varieties.A five-year field experiment was conducted to reveal the reasons for the differences in lint yield and fiber quality across three Bt cotton varieties with different yield formations from 2013 to 2017.The lint yield of Jiman 169(the average yield from 2013-2017 was 42.2 g/plant)was the highest,i.e.,16.3 and 36.9%higher than Lumianyan 21(L21)and Daizimian 99B(99B),respectively.And the differences in boll weight among the three cultivars were similar to the lint yield,while the others yield components were not.So the increase in lint yield was mainly attributed to the enlargement in boll weight.However,the change in fiber quality was inconsistent with the lint yield,and the quality of L21 was significantly better than that of Jimian 169(J169)and 99B,which was caused by the diversity of boll spatial distribution.Compared with 99B,the loose-type J169 had the highest number of large bolls in inner positions;the tight-type L21 had a few large bolls and the highest number of lower and middle bolls.And approximately 80.72%of the lint yield was concentrated on the inner nodes in Jiman 169,compared with 77.44%of L21 and 66.73%of 99B during the five-year experiment.Although lint yield was significantly affected by the interannual changes,the lint yield of J169 was the highest and the most stable,as well as its yield components.These observations demonstrated the increase in lint yield was due to the increase in boll weight,and the large bolls and high fiber quality were attributed to the optimal distribution of bolls within the canopies.

Bt cotton influencing enzymatic activities under varied soils

The enzymatic activity was evaluated under both Bt and non-Bt systems in varied soil type. The study was conducted during the 2010 wet season (July to December) in a net-house at the Institute of Agricultural Sciences of Banaras Hindu University. It was carried out on three different soil ordersi.e.entisol, inceptisol and alfisol. Bt cotton (cvNCS-138) and its non-transgenic isoline (cvNCS-138) were grown until maturity. A no crop pot was maintained with three replications for all the three soil orders. Study design was a factorial experiment under a completely randomized block design with three replications. The study concludes that soil under Bt cotton cultivar produced significantly higher amount of phosphatase activity than both nonBt and no crop treatments at three growth stages. And the value decreased as the crop growth period advanced. The interaction effect between soil type and Bt-crop was found to be significant in different growth stages throughout the growing season. Results from the study revealed that a significant reduction (9.4%) of the dehydrogenase activity and soil respiration (5%) in the rhizosphere of Bt cotton over non-Bt isoline.

Amino acids application enhances flowers insecticidal protein content in Bt cotton

Background:Low insecticidal protein expression at reproductive organs affect insect resistance in Bt transgenic cotton.In order to enhance flower insecticidal protein expression,the conventional cultivar Sikang1(S1)and the hybrid cultivar Sikang3(S3)were used as experimental materials;the applications of selected 5 types of amino acids and 21 types of amino acids were sprayed on the flowers in 2016 and 2017 cotton growing seasons.Results:The flower Bt protein contents increased significantly under the two amino acid treatments in both cultivars,the Bt protein concentration increased by 15.2 to 25.8%compared with the control.However,no significant differences were detected between the two treatments of amino acid application.Increased amino acid and soluble protein contents,enhanced GPT,GOT,protease,and peptidase activities were observed under the amino acid application at the flowering stage.Conclusions:These results suggest that exterior application of the amino acids treatments could bolster the flower insecticidal protein expression.

Effects of NaCl stress on the biochemical substances in Bt cotton as well as on the growth and development and adult oviposition selectivity of Helicoverpa armigera

Background:Recently,due to the development of food security strategies,cotton has been planted in inland saline-alkali dry soils or in coastal some saline-alkali soils in China.Under the condition,to comprehensively prevent and control Helicoverpa armigera in cotton fields with saline-alkali soils,it is important to study the larval growth and development of H.armigero and to study adult oviposition selectivity in H.armigera adults that feed on NaCI-stNaCled cotton plants.Results:In this study,Bt cotton GK19 was used for the experimental group and its nontransgenic parent Simian 3 was used for the control to study the effects of biochemical substances in cotton as well as larval growth and development and adult oviposition selectivity of H.armigera.The experiments were performed by growing cotton indoors under NaCl stress at concentrations of 0 mmol-L-1,75 mmol-L-1 and 150 mmol-L-1,respectively.The results showed that the expression of Bt protein was significantly inhibited for NaCI-stressed Bt cotNaClThe content of soluble protein and K+ in the leaves of cotton were decreased,while the content of gossypol and Na+were increased.In addition,the 5th instar H.armigera larvae exhibited shorten the life span in a 13-day trial period.Under enclosure treatments and at different female densities,the adult oviposition of H.armigera decreased on high NaCI-stressed nontransgenic coNaCl,while the oviposition on Bt cotton tended to first increase but then decrease under low,moderate and high NaCl stress treatments.Conclusions:Under certain content ranges of NaCl stress treatments,larval of H.armigera growth and development,and adult oviposition were no significant difference in the change for a certain period.However,under high NaCl stress,larval growth,development and adult oviposition were affected,which may provide insights for the prevention and control of H.armigero for Bt cotton in saline-alkali soils.

Chinese-version Bt Cotton: How to Get the Benefi ts from IPR

Chinese scientists started to fully synthesize the Bacillus thuringiensis (Bt) Cry1A gene in 1991. By the end of 1992, Biotechnology Research Institute of Chinese Academy of Agricultural Sciences (BRI-CAAS) success-

Azotobacter inoculation can enhance the resistance of Bt cotton to cotton bollworm, Helicoverpa armigera

The rising trend in the cultivation of Bacillus thuringiensis (Bt) transgenic crops may cause a destabilization of agroecosystems, thus increasing concerns about the sustainability of Bt crops as a valid pest management method. Azotobacter can be used as a biological regulator to increase environmental suitability and improve the soil nitrogen utilization efficiency of crops, especially Bt cotton. A laboratory test investigated effects on the development and food utilization of Helicoverpa armigera fed with different Cry1Ab/Cry1Ac proteins and nitrogen metabolism-related compounds from cotton (transgenic variety SCRC 37 vs non-Bt cotton cv. Yu 2067) inoculated with Azospirillum brasilense (Ab) and Azotobacter chroococcum (Ac). The findings indicate that inoculation with Azotobacter significantly decreased the partial development and food utilization indexes (pupal weight;pupation rate;adult longevity;fecundity;relative growth rate, RGR;efficiency of conversion of digested food, ECD;and efficiency of conversion of ingested food, ECI) of H. armigera fed on Bt cotton, but contrasting trends were found among these indexes in H. armigera fed on non-Bt cotton inoculated with Azotobacter, as a result of differences in Bt toxin production. Overall, the results showed that inoculation with Azotobacter had negative effects on the development and food utilization of H. armigera fed on Bt cotton, leading to enhanced target insect resistance. Presumably, Azotobacter inoculation can be used to stimulate plant soil nitrogen uptake to increase nitrogen metabolism-related compounds and promote plant growth for Bt and non-Bt cotton, simultaneously raising Bt protein expression and enhancing resistance efficacy against cotton bollworm in Bt cotton.

Enhancing boll protein synthesis and carbohydrate conversion by the application of exogenous amino acids at the peak flowering stage increased the boll Bt toxin concentration and lint yield in cotton

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.