Transformation Of Rice With Amaranth AmA1 And HlF Gene

Rice (Oryza sativa L.) is one of the most important food crops. The nutritional quality of rice has great influence on human health and rice production. The protein, essential amino acid, microelement are the nutritional qualities of rice that are maily concernted.The contents of essential amino acids such as lysine, serine, methione and tryptophan, etc. are relatively low in rice grain, which decreases the utilization effiecency of protein and the nutritional value. Thus, it is necessary to increase the contents of essential amino acid content of rice grain so that the nutritional value can be improved. The AmA1 is a protein isolated from the seed of Amaranthus L. that posesses blanced essential amino acids, the contents of all eight essential amino acids are above the ideal protein standard that is recommended by the FAO/WHO. Also, this protein is friendly for human digestion and absorption. These properties have made the AmA1 a promising protein in the improvement of the nutritional quality of the crop seed protein. Anther problem of the nutritional quality of the rice grain is that the iron content is relatively low as well. The human lactoferrin (hLF) has a number of biochemical functions that are capable of not only improving iron nutrition but also bearing broad spectrum of antibacteria and adjusting the immune response of human body, etc. The application of hLF to rice will increase the biological effectiveness of iron in human body.The improvement of rice nutritional quality with conventional breeding methods has made quite a lot of progress. However, due to the inadequacy of rice germplasms and reproductive isolation, the application of conventional breeding methods has only limited success. The development of genetic engineering technique provides a feasible method for improving the nutritional quality of rice grain. In an attempt to improve the compositon of essential amino acids of rice grain and increase iron nutrition, the AmA1 and hLF gene were transformed to rice with transgenic technique in order to express the proteins in rice seed with high efficiency and specificity in this study. The main results are as follows:1.In order to lead AmA1 and hLF expressed and accumulated in rice seed with high efficiency and specificity. the two promoters of rice glutelin gene(GluB-1 and Gt1) and one promoter of oil membrane gene ole18 were cloned in this study according to the nucleotide sequences of the promoter of rice glutelins that are publicly available. The above three pomoters and the constitutive promoter CaMV 35S were ligated with reporter gene GUS and transformed to rice respecitively to determine the expression intensity and specifictiy. The results of sequence analysis and GUS assay of transgenic rice showed that: (1) The promoter sequenceses of Gt1 and ole18 cloned from rice cultivar"Nipponbare"were identical with the reported sequences. The promoter sequence of the GluB-1 cloned from rice cultivar "TG9"has a homology of 97% with the reported sequence. The analysis of promoter prediction software NNPP and cis-acting element and transgenic rice experiment indictated that the promote could guide the GUS gene to express specifically in rice embryo and the nucleotide sequence difference did not affect its function in this study. (2) The expression intensities of the GUS gene with regulation of Gt1 and GluB-1 did not show much difference, but they were both higher than that of the CaMV35S. The Gt1 and Ole18 both have stringent tissue specificity of expression. The Gt1 guided the specific expression of GUS in rice embryo and the Ole 18 guided the specific expression of GUS in rice embryo and seed capsule.2.The OFR sequence of the AmA1 was cloned from the seed of Amaranthus L. cultivar"Amaranthus hypochondriacus L.NO1". The sequence analysis showed that the cloned sequence isessentially identical with the reported sequence. There was only one nucleotide difference but the amino acid coding was the same. The molecular chaperonevectors pBB540 and pBB542 were applied to assist the soluble expression of the pET28a-AmA1 in E. coli and the target protein was purified with Ni-NTA affinitychromatography. The results indicated that the IPTG could induce the production of the recombinant protein,of which the moleclar weight was consistent with the the oretical molecular weight of 37kDa. Thus, the utilization of the molecular chaperone vector could significantly improve the the soluble expression of the recombinant protein AmA1.3.The reported method of wheat rapid DNA extraction was simplified at some steps and then applied to rice in this study. The results showed that rice genomic DNA extracted by this simplified method was intergal and the PCR amplification using it as template was stable and reliable, showing no significant difference from plant genomic DNA extraction kit method. Furthermore, the whole extraction procedure is simple, takes less time(only 5~6 min per sample on average), and requires a small amount of plant sample about 5~10mg. Therefore this method is suitable for the large-scale PCR detection of transgenic rice.4.The plant expression vectors with two T-DNA and dual MARs were constructed, which were PCDMAR-pGt1-AmA1-hpt and and PCDMAR-pOle18-AmA1-hpt under the control of Gt1 promoter and Ole18 promoter respecitively, and transformed to MH86 and TG9 with agrobacterium mediated method. The PCR andSouthern blot analysis showed that the AmA1 genes in the two vectors were integrated to the rice genome. The Western blot results showed that the Gt1 promoter and Ole18 promoter could guide the correct expression of AmA1 in rice seed. The assay of the amino acid content in the grain of T1 transgenic rice showed: (1) seven amino acids contents (accounting for the total dry weight) in the 9 lines in which the AmA1 expression was regulated with Gt1 promoter were higher than those of the control to various degrees, the largest of which was 44.44%. The contents (accounting for the percentage in the total amino acids) of three essential amino acids (lysin,threonine and methionine) in the lines were significantly higher than those of the control, with the maximun increase of lysine 5.58%, threonine 3.12% and methionine5.58%. (2) seven amino acids contents (accounting for the total dry weight) in the 5 lines in which the AmA1 gene expression was regulated with Ole18 promoter were higher than those of the control to various degrees, the largest of which was 15.56% and there was only one strain that hasa 4.4% increase of lysine content in the total amino acids. The above results suggested that the AmA1 gene could express specifically in rice embryo under the regulation of Gt1 promoter and the contents of the three essential amino acids were increased in a notable extent. 5.Under the premise of not altering the amino acid sequence, codon usage of native hLF and AmA1 genes are optimizied according to rice preferred codons. The sequence elements that affect the efficiency of gene transcription, translation and stability of mRNA are readjusted or removed purposefully for efficient plant expression. The 5′and 3′ends of the gene are modified at the same time. The main results are listed as follows:(1) The optimized and modified hLF gene shares 76.3% nucleotide sequence similarity to the wild hLF gene in the coding region. Of the 692 codons for the mature peptide of hLF gene, 431 codons are changed. The ration of total G+C content and G+C cotent of bases at the third position in codons are increased from 53.82%, 59.88% of the wile type to 56.28% ,66.38% of the modified. Three of PPSS (potential polyadneylylation signal sequence) and 3 of A+T rich regions are removed entirely. Then for the optimized outer-gene code, we strip the rice GluB-1 gene signal peptide sequences in the 5′end,KDEL and double terminators sequences in the 3'the end,respectively. (2) The optimized and modified AmA1 gene share 76.0% nucleotide sequence similarity to the wild AmA1 gene in the coding region. Of the 305 codons for the mature peptide of AmA1 gene, 192 codons are changed. The ration of total G+C content and G+C cotent of bases at the third position in codons are increased from 34.43% and 29.51% of the wild type to 49.95%, 71.48% of the modified. The number of PPSS,ATTTA sequences and A+T rich regions are decreased from 17,3 and 2 to 5, 0 and 1, respectively. Then for the optimized outer-gene code, we strip the rice Gt1 gene signal peptide sequences in the 5'end.6. The optimized hLF ( named omhLF) and AmA1 ( named omAmA1) gene are synthesized. Then the native and optimized hLF,AmA1 gene are transferred into rice cultivar"TG9"respectively for the purpose of comparing their expression level difference in rice. 178 PCR-positive line are obtained. Meanwhile, we construct a plant expression vector containing omhLF and omAmA1 gene and transfers it into rice cultivar"SH527"by agrobacterium-mediated method. The result of PCR detection shows that fragment of omhLF and omAmA1 gene with expected size are amplified from 34 transgenic lines. Due to be at tillering stage of T0 generation, further molecular identification and quality analysis for these lines will be conducted in the next time.