Study On Genetic Transformation Of Rice With High-lysine Gene CFLR And Amaranth Balanced Protein AMA1Gene

As the first limiting amino acid in the rice protein,lysine’s content is directlyrelated to how much human body can effectively use the rice protein,there are about4.0g/16gN lysine in the general rice varieties,and if the content can not be improved,the protein can only be use58%, and the remaining42%is wasted.CFLR,one gene coding high-lysine protein,content up to21.2%,isolated from thepepper’s mature pollen, is known as the highest lysine-rich of the natural proteingene. The AmA1is a balanced protein, is separated from the immature Amaranthus L.seeds that contains eight kinds of essential amino acids content in excess of the FAO/WHO recommended standards of the ideal protein.In addition, the CFLR protein andAmA1protein used in genetically modified are friendly for human digestion andabsorption. Therefore, these two protein genes can play an important role in the cropseeds’ protein nutrition inprovement.In this study, we transferred the optimized gene CFLR and gene AmA1to therice at the same time, highly expressed, and improvement of the essential amino acidsin rice is expected. The main findings are as follows:(1) CFLR gene is optimizied under the premise of the amino acid sequence doesnot change, according to rice codon preferences,and considered the sequence elementsthat affect the efficiency of gene transcription, translation and stability of mRNA arereadjusted or removed purposefully.Compared the optimized CFLR nucleotidesequence coding region with the original consistency is up to75.3%,G+C contentincreased from40.3%to50.9%,in the gene interaction, the number between thepurine conversion and the pyrimidine interconversion is98,and the Reversed swap ofthe purine and pyrimidine is71.(2) Constructed the plant expression vector PCDMAR-AmA1-CFLR-hpt withdouble genes CFLR and AmA1at the same time,it contained two spereate T-DNAregions,and the double genes AMA1and CFLR and their promoter pGt1and pGluB-1is carried by one T-DNA region, these two promoters are specificity express in riceendosperm,the other T-DNA region carried the selectable marker gene hpt. (3) Transformed the double T-DNA plant expression vector PCDMAR-AmA1-CFLR-hpt into the rice quality varieties of “Taijing9” by theAgrobacterium-mediated method, and we obtained76“Taijing9” T0generationtransgenic rice by the selection of culture medium,14of which were positive by PCRtesting.(4) We measured the content of total protein and amino acid in the rice seeds ofthe TOgeneration. Four rices of the total protein content are increased significantlycompared with the CK,the maximum was43.79%, the minimum was15.13%. Alleight rices’ lysine、methionine and threonine relative content were increased comparewith theCKthreonine increased by3.3%to8.93%,methionine increased by22.90%to117.33%,lysine increased by0.03%to13.43%.