| ObjectivesThe nutrients’composition analysis, the iron bioavailability of genetically modified rice with human lactoferrin gene (hLF) and the effect on iron deficiency anemia in rats that feed by this rice was evaluated; the allergenicity and toxicity was observed to assessed the edible safety of the recombinant human lactoferrin (rhLF) isolated and purified from hLF rice; the inhibitory effect of bacterium of rhLF in vitro and in vivo in mice with Salmonella typhimurium infection was observed to evaluate the special efficacy of rhLF; the nutritional value of two varieties of genetically modified wheat expressing GmDREB1/TaDREB4genes with drought-resistance was assessed.Methods1The nutrients’ composition analysis and efficacy assessment of genetically modified rice with human lactoferrin gene (hLF rice)1.1Nutrients’ analysis and the determination of hLF Protein, carbohydrates, fat, vitamin, minerals etc in hLF rice and their parental rice was determined according to the National Standards of China. ELISA was used to determine hLF in the transgenic rice. 1.2The iron bioavailability study In vitro digestion/Caco-2cell model was used to determine the absorption rate of iron in genetically modified rice with human lactoferrin gene and its parental rice.1.3Study of improving the iron deficiency anemia in rats Weanling female Wistar rats weighing45-55g were induced by iron deficient diet. The rats was then randomly divided into eight groups based on their body weight and the concentration of hemoglobin:ferrous sulfate group, AIN group, control rice with high dose, control rice with middle dose, hLF rice with high dose, hLF rice with middle dose, bovine lacotoferrin and the model control, respectively. Each group was fed different feed. Each rat was weighed once a week and its diet intake was weighed twice a week. At the begaining, in the middle and at the end of the experiment, blood of each rat was taken to determine the blood routines.2Isolation and purification, edible safety and efficacy assessmentof recombinant human lactoferrin2.1Isolation and purification from transgenic rice and determination of rhLF After isolatated from transgenic rice via saturated ammonium sulfate precipitation, rhLF was purified through CM Sepharose FF-exchange chromatography and molecular sieve chromatography Sephadex G25. Westernblot was used to examine the purified rhLF; Gel image analysis software was used to analyze the purity of rhLF; Mass-spectrography was used to determine the molecular weight, protein weight and the c-terminal amino acid sequence; isoelectric focusing electrophoresis was adopted to determine the isoelectric point of this protein.2.2Bioinformatics analysis and serum allergenic study The amino acid sequence of rhLF was analyzed in known allergensin four databases, such as ailergenonline, SDAP, Blast and Sortaller, which are widely used in the world at present. The primary methods of evaluation were searching for "80amino acid alignments with greater than35%identity by FASTA" and "8amino acid exact matches". As the serum screen, sera from26patients with allergic including12acarid-allergic subjects,4soybean-allergic,6peanut-allergic and4cat-allergic individuals in this study were conducted to do Immunoblots.2.3Digestive stability and heat stability Preparation of simulated gastric and intestinal fluid to establish the in vitro evaluation method on digestive stability and heat stability of rhLF. With SDS-PAGE method, digestive stability of rhLF was assessed in vitro in simulated gastric and intestinal fluidat time Os,15s,30s,60s,2m,4m,8m,15m,30m and60min and heat stabilityof rhLF was determined by heat treatment at the time of5m,10m,15m,20m,30m,45m,60m,90m and120m.2.4Acute oral toxicity study The test was conducted according to the National Standards of China GB15193.3-2003. Sixty6week old Kunming strain mice weighting17-22g was used. After acclimatization for7days, the mice were randomized for three groups:rhLF from rice group, BSA control, and deionized water, ten male rats and ten female rats for each group.The maximum tolerated dose method by gavage at a time was adopted. The average gavage volume was20ml/kg-BW for eacha mouse, which is about0.4ml/20g. After gavage, mortality and clinical signs were observed. Food intake was recorded twice every week. Body weight was measured on days0,3,7and14. All animals were necropsied at the end of experiment. Absolute and ratio of organ vs body weight was recorded in the following organs:heart, kidney, liver, spleen and sex glands.2.5Antibacterial activities in vitro Using broth microdilution method, the final concentration of rhLF from transgenic rice and milk of transgenic cows was adjusted under different concentrations of rhLF (0.25,0.50,1.00,2.00,4.00, and5.00mg/ml). Then the inhibitory effects of bacteria was observed on5kinds of gram-negative bacteria including P.AeruginosaATCC27853, Enterobacter sakazakiiATCC51329, Salmonella typhimurium ATCC50333, Escherichia coli ATCC25922and Shigella CMCC51592; and three kinds of gram-positive bacteria including Staphyloccocusaureus ATCC29213, Listeria monocytogenes CMCC54004and Bacillus cereus ATCC11778. OD620of the bacteria was monitored on the time Oh,2h, 4h,6h,9h,18h and24h afrer culture. Bacterial cultures without hLF were used as positive controls, and uninoculated BHI broth was used as a negative control.2.6Effect of rhLF on Salmonella ser. Typhimurium infection in miceDetermination of LD506-8week old female Balb/c mice weighting17-20g was used. After acclimatization for5days, the mice were randomized for four groups to determin LD50of Salmonella ser. Typhimurium ATCC50333using oral gavage method. Each group included10mice. Then one of five and one of ten of the LD50was adopted for the infection dose.Effect of rhLF on Salmonella ser. Typhimurium infection in mice The similar mice used in the LD50test were randomized for five groups with each group12:L ST (low concentration of Salmonella ser. Typhimurium), L ST+rhLF from transgenic cows, H ST (high concentration of Salmonella ser. Typhimurium); H ST+rhLF from transgenic cows, H ST+rhLF from transgenic rice, respectively. An oral gavage of rhLF20mg/ml was given to the mice2hours before infection. After infection by oral gavage, rhLF was given everyday and lasted for7days. The control group was given the same volumn of PBS. The mortality and clinical signs were monitored. Body weight was measured everyday. Seven days after infection, the blood of mice was obtained from heart punctutre for determination of bacteriaemia; the liver and spleen was obtained for analysis of bacterial count; the liver, spleen, kidney and intestine was obtained for pathology observation.3Nutritional assessment of two new varieties of genetically modified wheat expressing GmDREBl/TaDREB4genes with draught-resistance3.1Nutrients’ analysis Protein, carbohydrates, fat, vitamin, minerals and18kinds of amino acid in genetically modified wheat and their parental wheat was determined according to the National Standards of China.3.2Protein efficiency ratio tests According to the ministry of agriculture line standard (the standard of project approval in2012), the28days feeding test was used to assss the protein efficiency ratio (PER) of two new varieties of genetically modified wheat. Eighty Wistar rats, weighing55-65g, were randomly divided into four groups, with ten male rats and ten female rats for each group. By taking maximum protein addition as the principle, protein content of each diet was formulated up to10%, while the insufficient part was supplemented by casein. All the other ingredients were added according to the formulation recommended by AIN-93G. Rats in each group were given Parental control wheat diet, the transgnic wheat expressing GmDREBl gene diet, the transgnic wheat expressing TaDREB4gene diet and casein diet, respectively. Each rat was weighed once a week and its diet intake was weighed twice a week. At the end of the experiment, blood of each rat was taken to determine the blood routines and blood biochemistry. All animals were necropsied at the end of experiment. Absolute and ratio of organ vs body weight was recorded in the following organs: heart, kidney, liver, spleen and sex glands. Then the PER and adjusted PER of each group was calculated and compared.ResultsIThe nutrients’ composition analysis and efficacy assessment of genetically modified rice with human lactoferrin geneThe results of nutrients’ analysis and the determination of hLF showed that, there was no statistical difference between hLF rice and its parental rice in main nutrients (P>0.05). Except one or two elements such as copper and phosphor, there was no significant difference existed in most of minerals and vitamins between the two groups. The average content of hLF in the seed dry weight was about0.5%.The results of iron bioavailability from the in vitro/Caco-2cell models showed that there was no statistical difference between hLF rice and its parental rice on the asorption rate of iron (P>0.05). The results of effect on the iron deficiency anemia in rats showed that there was no statistical difference in the body weight increase and feed intake among the groups except the model control group in the middle and at the end of the experiment (P>0.05). The blood routines showed that the Hb, RBC and other haematological index of groups including ferrous sulfate, AIN, hLF rice with high dose, hLF rice with middle dose and bovine lacotoferrin was obviously increased when compared with the model control group (P<0.05). However, the Hb of both the two control rice groups was also increased (P<0.05). The Hb of hLF rice with high dose, hLF rice with middle dose and bovine lacotoferrin group was lower than that of two control rice groups (P<0.05). The RBC of hLF rice with high dose, hLF rice with middle dose was similar between the hLF rice with high dose, hLF rice with middle dose and the two control rice groups. While the RBC of bovine lacotoferrin group was lower than that of the two control rice groups (P>0.05).2Isolation and purification, edible safety and efficacy assessment of recombinant human lactoferrinThe results of the isolation and purification and basic characterization determination showed that the rhLF was obtained at a higher purity (about90%) after isolatated rhLF from transgenic rice via saturated ammonium sulfate precipitation, and then purified it through CM Sepharose FF-exchange chromatography and molecular sieve chromatography Sephadex G25. After Coomassie blue staining, Westernblot and mass spectrometer analysis, it was identified as the purpose protein with the molecular weight of approximately79kDa.The isoelectric point was8.02; the fifteen amino acid sequence of the c-terminal amino acid sequence was similar to the native human lactoferrin.The peptide fingerprint of the protein showed it was matched to the the native human lactoferrin.The results of bioinformatics analysis showed that there was high-level similarity between and some known allergens, such as lactotransferrin and ovotransferrin in the amino acid sequence. The results of serum allergenic study showed that there was no cross-reactivity in most of serum, while there were two positive reactions from acarid-allergic serum.The two studies indicated that rhLF may have allergenic potential.The results of digestive stability and heat stability of rhLF showed that rhLF was digested within30s in simulated gastric fluid, which indicated that it was instabile in the gastric fluid, while it was stabile in the intestinal fluid. As to the heat treatment, rhLF was degraded within15m, which indicated instabile when heating. The LD50of rhLF was more than6.68g/kg.The results of acute oral toxicity study showed that there was no abnormal performance in the male and female mice. There was no significant difference in the weight, food intake and absolute and ratio of organ vs body weight (P>0.05).The result of antibacterial activities in vitro showed that both rhLF from transgenic rice and milk of transgenic cows could inhibit eight kinds of bacteria when compared with their own positive control group. In gram-negative bacteria, both of rhLF could obviously inhibit PAeruginosa, Enterobacter sakazakii and Salmonella typhimurium, while both of them could inhibit Escherichia coli and Shigella at higher concentration.In gram-positive bacteria, both of rhLF could obviously inhibit Staphyloccocusaureus and Bacillus cereus. rhLF from transgenic cows seemed more sensitive to Listeria monocytogenes than that from transgenic rice.The result of antibacterial activities in vivo showed that after Salmonella ser. Typhimurium infection for seven day, the mice of the groups given by rhLF performed better than that of the control groups either in low or high concentration of ST.There was no significant difference in the body weight and motality of mice between control group and rhLF group (P>0.05). However, the wet weight of spleen, the ratio of spleen vs body, the infection rate in the spleen, the bacterial count in the liver and spleen of mice of L ST+rhLF from transgenic cows was significantly less than that of the L ST control group(all P<0.05). The level of bacteria in the blood, liver and spleen of H ST+rhLF from transgenic cows and H ST+rhLF from transgenic rice was also much lower than that of the H ST control group, while the differences did not show any stasistical meaning.3Nutritional assessment of two new varieties of genetically modified wheat expressing GmDREBl/TaDREB4genes with draught-resistanceThe results of nutrients’ analysis showed that there was no statistical difference in the main nutrients, minerals and vitamins between the two kinds of genetically modified wheat with draught-resistance and the parental contrl wheat (P>0.05) except one or two element such as fat and tryptophan. There was neither no significant difference existed in17amino acid among the three groups (P>0.05).The results of the PER test showed that the general situation was well and the behavior of rats was normal in all groups. There was no obvious differences in the body weight and the feed intake for the four weeks in the two varieties of transgenic wheat and the parental control wheat (.P>0.05). There was some indexes of blood routines,blood chemistry and ratio of organ vs body weight of the two genetically modified wheat was different from that of PR group or the casein group (P<0.05), while all the deviation were belong to the normal reference ranges. There was no significant difference in PER between the two genetically modified wheat and the PR group(P>0.05), which indicated that the PER of both transgenic wheat was equal to that of the parental control wheat. The PER of the two kinds of transgenic wheat was lower than that of the casein group(P<0.05), which indicated that the PER of both transgenic wheat was inferior to that of the casein group. After adjusted, the PER of transgenic wheat expressing GmDREBl gene, transgenic wheat expressing TaDREB4gene and the parental control wheat was0.76,0.75and0.67respectively.Conclusion1The nutrients’compositionanalysis and efficacyassessment of genetically modified rice with human lactoferrin gene (hLF rice) The content of rhLF in the transgenic rice was about0.5%, which was accordant with related reports abroad. There had not been able to observe the obvious effect on the iron bioavailability and the iron deficiency anemia of rats.2Isolation and purification, edible safety and efficacy assessment of recombinant human lactoferrinThe rhLF was obtained at a higher purity (about90%) through successful isolation and purification, which was similar to the native human lactoferrin after basic characterization determination. The bioinformatics analysis and serum allergenic study indicated that rhLF may have certain allergenic potential. rhLF from the transgenic rice, was instable in simulated gastric fluid and but stale in the simulated intestinal fluid. It was also easily degradated with heat treatment. The acute oral toxicity study indicated that rhLF was not toxic proteins factually. The result of antibacterial activities in vitro demonstrated that rHLF could have a wide spectrum to inhibit gram-negative bacteria and gram-positive bacteria. In vivo, rhLF from transgenic cows also could decrease the Salmonella typhimurium level in the body of mice, which indicated that rhLF may can prevent and alleviate the infection caused by bacteria.3Nutritional assessment of two new varieties of genetically modified wheat expressing GmDREBl/TaDREB4genes with draught-resistanceThere was no differences in the nutrients and PER between both transgenic wheat expressing GmDREBl/TaDREB4genes with draught-resistance and the control wheat. |
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