Safety Assessment Of Genetically Modified Rice With Lysine-rich Gene And Genetically Modified Rice With Human Lactoferrin Gene And Expression And Purification Of Exogenous Protein In Genetically Modified Rice With Cowpea Trypsin Inhibitor Gene

ObjectivesThrough nutrients'composition analysis and animal feeding experiments, the edible safety of genetically modified (GM) rice with lysine-rich gene and GM rice with human lactoferrin gene was assessed in this study. The impact of GM rice on the growth and development, digestibility and metabolism, subchronic toxicity and state of nutrition and health of animals were observed to assess the edible safety of the two types of rice in multi-aspects. In order to assess the edible safety of exogenous cowpea trypsin inhibitor (CpTI) protein in GM rice with CpTI gene, we utilized Escherichia coli prokaryotic expression system to recombine and expression the protein, which make it available for future assessment on toxicity and allergy.Methods1. Safety assessment of genetically modified rice with lysine-rich geneNutrients'analysis Protein, carbohydrates, fat, vitamin, minerals etc in genetically modified rice with lysine-rich gene was determined and compared with their parental rice (WT rice).In vivo digestibility of main nutrients in genetically modified rice with lysine-rich gene Eight healthy castrated male WZSPs, weighing 30-35kg, were individually housed in stainless steel cages. All WZSPs were surgically fitted with post-valve T-intestinal cannulas after 16-hour fasting control. Six WZSPs, which were well recovered from the surgery and with good appetite, were selected. A replicated latin square design was applied for GT3 rice, HL5 rice and WT rice comparison. Cr2O3 were added in diets as inert indicator, and the casein diet was served to determine the endogenous amino acid losses (EAL). Each diet was supplied for 7 days. During each of the last 3 days, ileal digesta was continuously collected by self-designed honeycomb ducts. Ileal digesta was freeze-dried and ground through a 0.250-mm mesh screen. Main nutrients in rice were determined and digestibilities of them were then calculated and compared. Amino acid scores (AAS) and protein digestibility corrected amino acid scores (PDCAAS) were used to assess the quality of protein in GM rice.Subchronic toxicity assessment (90-day feeding experiment) Sixty weanling Wistar rats, weighing 60-75 g, were randomly divided into three groups, ten male rats and ten female rats for each group. They were fed GM diet (HL5 group), Non-GM diet (WT group) and AIN-93G formula diet (AIN control), respectively. By taking maximum protein addition as the principle, protein content of each diet was formulated up to 20%, while the insufficient part was supplemented by casein. Each rat was weighed once a week and its diet intake was weighed twice a week. Blood of each rat was taken from eye plexus to determine the blood routines and blood biochemistry in the middle and at the end of the experiment. All the rats were sacrificed slaughtered at the end of the experiment, right femurs were taken to determined bone density. Routine pathological examination and organ coefficient for main viscera were also carried out.2. Safety assessment of genetically modified rice with human lactoferrin geneNutrients'analysis Protein, carbohydrates, fat, vitamin, minerals etc in genetically modified rice with human lactoferrin gene (hLF) were determined and compared with its parental rice (PR rice).In vivo digestibility of main nutrients in genetically modified rice with human lactoferrin gene (hLF rice) Six healthy castrated male WZSPs with T-intestinal cannulas were individually housed in stainless steel cages. A replicated latin square design was applied for hLF rice and its parental rice (PR rice). Cr2O3 were added in diets as inert indicator and the casein diet was served to determine the endogenous amino acid losses (EAL). Each diet was supplied for 7 days and during each of the last 3 days, ileal digesta was continuously collected and freeze-dried. Main nutrients in rice were determined and digestibilities of them were then calculated and compared. All pigs were sacrificed at the end of the experiment and dissected to inspect whether cannulation had caused intestinal abnormalities.Blood routines and blood chemistry were also determined. Amino acid scores (AAS) and protein digestibility corrected amino acid scores (PDCAAS) were used to assess the quality of protein in hLF rice and PR rice.Subchronic toxicity assessment (90-day feeding experiment) Eighty weanling Wistar rats, weighing 60-75g, were randomly divided into four groups, ten male rats and ten female rats for each group. They were fed GM diet (hLF high-dose group and hLF medium-dose group), Non-GM diet (PR group) and AIN-93G formula diet (AIN control), respectively. Each rat was weighed once a week and its diet intake was weighed twice a week. Blood of each rat was taken to determine the blood routines and blood biochemistry in the medium and at the end of the experiment. All the rats were sacrificed at the end of the experiment, right femur were taken to determined bone density. Routine pathological examination and organ coefficient for main viscera were also carried out.Nutritional assessment The protein efficiency ratio (PER) method was used to determine protein quality of PR rice and hLF rice by use of weanling Wistar rats. Sixty Wistar rats, weighing 55-65g, were randomly divided into four groups, 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 to 10%, 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 PR rice diet, hLF high-dose rice diet, hLF medium-dose rice diet and casein diet respectively. At the end of the experiment, blood of each rat was taken to determine the blood routines and blood biochemistry. PER of each group was calculated and compared.3. Expression and purification of cowpea trypsin inhibitor (CpTI) proteinFusion expression and purification Fusion expression vector pTWIN2-CpTI was constructed by cloning CpTI gene into vector pTWIN2. The fusion protein chitin-intein-CpTI was induced by IPTG, and the CpTI protein was separated and purified by chitin column chromatography, temperature and pH variation. Fusion expression vector pPAL7-CpTI was constructed by cloning CpTI gene into vector pPAL7. The profanity tag of expressed fusion protein could be removed by affinity chromatography purification, and the CpTI protein was therefore obtained and purified.Non-fusion expression and purification The tag of PET41EK/LIC vector was removed, and non-fusion expression vector PET41EK-CpTI was then constructed by cloning CpTI gene into tag-removed PET41EK/LIC vector. The condition of expression and ion-exchange purification was optimized to obtain CpTI protein without any extra amino acid residue as much as possible.Results1. Safety assessment of genetically modified rice with lysine-rich geneThere was no statistical difference between genetically modified rice with lysine-rich gene (GT3 rice and HL5 rice) and its parental rice (WT rice) in main nutrients.The results of digestibility of main nutrients in vivo showed that there was no statistical difference between GM rice with high-lysin gene and their parental rice on main nutrients'digestibility (P>0.05). The GM rice with lysine-rich gene did not interfere normal digestibility and metabolism of protein/amino acids and other nutrients, and it did not interfere the growing, development and metabolism of animals either. The AAS of GT3 rice, HL5 rice and WT rice were 47.82%,65.11%,56.44% respectively, while their PDCAAS were 53.45%,67.49%,58.70% respectively.The results of subchronic toxicity assessment showed that there were no statistical difference between GM rice with high-lysin gene group (HL5 group) and AIN control group in terms of diet intake and bodyweight. Although some indexes of blood routines, blood chemistry, organ coefficient and bone density were significant different from WT group or AIN control group, majority of the indexed were within normal range reported by other researchers. No obvious abnormality was found in pathological examination.2. Safety assessment of genetically modified rice with human lactoferrin geneExcept for lactoferrin, lysine, iron, there was no statistical difference between genetically modified rice with human lactoferrin gene (hLF rice) and its parental rice (PR rice) on main nutrients.The results of digestibility of main nutrients in vivo showed that there was no statistical difference between hLF rice and PR rice on main nutrients'digestibility (P>0.05). hLF rice did not interfere normal digestibility and metabolism of protein/amino acids and other nutrients, and it did not interfere the growing, development and metabolism of animals either. The AAS of hLF rice and PR rice were 50.27%,53.24% respectively, while their PDCAAS were 54.06%,52.66% respectively.The results of subchronic toxicity assessment showed that there were no statistical difference hLF rice (high-dose and medium dose) group and AIN control group in terms of diet intake, bodyweight and calcium concentration in blood. Although some indexes of blood routines, blood chemistry, organ coefficient and bone density were significant different from PR group or AIN control group, majority of the indexed were within normal range reported by other researchers. No obvious abnormality was found in pathological examination.The results of nutrition assessment showed that some indexes of blood routines, blood chemistry and bone density were significant different from PR group or AIN control group. In terms of protein efficient ratio (PER), hLF high-dose group was significantly higher than PR group, and hLF medium-dose group was significantly higher than the other three groups. The corrected PER of PR group, hLF high-dose group and hLF medium-dose group was 1.97,2.50 and 2.73 respectively, which indicated that the hLF rice was easier to be utilized and adsorption than PR group and the medium dose was easier to be utilized and adsorption than high dose.3. Expression and purification of cowpea trypsin inhibitor (CpTI) proteinCpTI fusion protein with trypsin inhibitor (TI) activity was obtained by pTWIN2-CpTI expression system, however, target CpTI protein could not be separated and purified by procedures of chitin choromatography, pH value and temperature variation. CpTI fusion protein with TI activity was also obtained by pPAL7-CpTI expression system, and target CpTI protein was obtained through affinity chromatography. The yield of pPAL7-CpTI expression system was 1.67mg/L induced bacteria liquid.CpTI protein with TI activity was obtained through non-fusion protein expression and ion-exchange purification optimization. The purity was above 95% and its yield was improved to 15mg/L induced bacteria liquid. The CpTI protein obtained in this study was consistent with natural CpTI protein in terms of TI activity, molecular weight and immunogenicity.Conclusions1. Safety assessment of genetically modified rice with lysine-rich geneCompared with the parental rice (WT rice), the main nutrient composition, digestibility of main nutrients in vivo and subchronic toxicity of GM rice with lysine-rich gene were studied. The results showed that there were no statistical differences between GM rice and WT rice in nutrients composition. The GM rice did not interfere normal growing, development and metabolism of experimental animals. No side effect was found in the subchronic toxicity assessment. AAS and PDCAAS of HL5 rice was higher than that of WT rice which indicated that the protein quality of HL5 rice might be slightly superior to WT rice. Significantly difference was found in some indexes in animal experiment, but all indexed values were almost within normal range.In conclusion, no side effect of GM rice with lysine-rich gene was found, and the edible safety of GM rice was equal to its traditional PR rice according to this study.2. Safety assessment of genetically modified rice with human lactoferrin geneCompared with the parental rice (PR rice), the nutrient composition, digestibility of main nutrients in vivo, subchronic toxicity and nutritional quality of GM rice with human lactoferrin gene (hLF rice) were studied. The results showed that there were no statistical differences between hLF rice and PR rice in nutrients composition except for lactoferrin, lysine and iron. The GM rice did not interfere normal growing, development and metabolism of experimental animals. No side effect was found in the subchronic toxicity assessment. AAS and PDCAAS of hLF rice were higher than that of PR rice, moreover, the PER of hLF rice was significantly higher than that of PR rice which indicated that the protein quality of hLF rice might be superior to PR rice. Significantly differences were found in some indexes in animal experiment, but all the values were almost within normal range.In conclusion, no side effect of hLF rice was found, and the edible safety of GM rice was equal to its traditional PR rice according to this study. The protein quality of hLF rice was superior to its traditional parental rice according to nutritional assessment.3. Expression and purification of cowpea trypsin inhibitor (CpTI) proteinThe exogenous protein of GM rice with cowpea trypsin inhibitor was expressed and purified by optimizing fusion and non-fusion prokaryotic expression system. The condition of induction and purification was studied, and the method of prokaryotic expression and purification for CpTI protein was optimized. The purity of CpTI obtain in this way was up to 95%, and the yield reached 15mg/L induced bacteria liquid. The method is very easy to operate and has less losses of target protein. And the CpTI protein acquired by this method laid a good foundation for toxicity and allergenecity assessment.