Enhancing Free Lysine Content In Rice By Metabolic Engineering

Plants are the primary source of proteins consumed by humans and livestock. However, most plant proteins are nutritionally unbalanced, because they are deficient in certain essential amino acids. In general, cereal proteins are low in lysine and tryptophan. Rice (Oryza sativa L.), one of the leading food crops and the staple food of over half the world's population, is a very good and relatively cheap source of energy and protein. However, like other cereals, rice proteins are nutritionally incomplete due to their deficiency in threonine, tryptophan, especially lysine. Based on our understanding the lysine biosynthetic and catabolic pathway, two apporaches could be carried out to enhance free lysine content in rice. One is to increase the rate of lysine synthesis by bypassing the feedback regulation in lysine biosynthetic pathway by expressed the genes encoding E. coli feedback- insensitive aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). Another approach is to down-regulate the rate of lysine catabolism by designing antisense or RNAi constructs to inhibitate the expression of the LKR/SDH (lysine- ketoglutaric acid reductase and saccharopine dehydrogenase) bifunctional enzymes, which controlled the rate of lysine degradation.In present study, by using the transgenic rice plants developed previously containing different target genes involved in lysine synthesis and catabolism described as above, the main purpose is to analysze and compare the effect of different target transgenes on enhancing lysine content in rce seeds, as well as to study the effect on rice growth, development and grain quality. The main results were showed as followings.1. To understand the expression pattern of rice LKR/SDH gene the GUS activity was carefully measured and compared among different transgneic rice plants containing GUS fusion gene controlled by the promoters with different length of rice LKR / SDH gene. The results showed that the LKR/SDH promoter with a 3.9 kb length upstream of translation initiation codon ATG could drive the expression of GUS gene in endosperm, leaf and stem of transgenic rice plants, and prefer a certain endosperm-specific expression. It was less of GUS expression level when driven by the 2.9 or 2.0 kb promoter with 5 'deletion than that by the 3.9 kb one, but the expression of GUS fusion gene controlled by a short length promoter, 1.6 kb upstream of ATG, was detected to be highest, and not show tissue-specificity. Therefore, within the region of 1.6 to 3.9 kb upstream of ATG, there might contain several cis-regulatory elements responsible for the fine expression of rice LKR/SDH gene.2. From the self-progeny of transgenic rice plants with different lysine metabolism-related genes, many homozygous transgenic lines were selected, and several transgenic lines without the hygromycin selectable marker gene were identified by both PCR ans Southern blot analyses. The results from Northern blots showed that the introduced AK and DHPS gene from E. coli, driven by either rice Gt1 or CaMV 35S promoter, could be highly expressed in developing seeds of most transgenic lines. In the endosperm of transgenic rice containing the LKR/SDH RNAi structure, the expression of endogenous LKR/SDH gene was significantly inhibited.3. The amino acid content in mature seeds was determined and compared among various types of transgenic rice containing different lysine metabolism-related genes. The results showed that, after compared with that of untransformed wild type, there was no or little change of free lysine content in the seeds of transgenic rice carrying both AK and DHPS genes. In the seeds of LKR/SDH-RNAi transgenic rice plants, the free lysine content was detected to increase by 5-7 times. After combined the three transgenes (AK, DHPS and LKR/SDH-RNAi) into the same rice plant, free lysine content in seeds siginificantly increased, and the maximum increase level was about 50 times over that of wild type. In transgenic lines with highly increased free lysine, the total lysine content was also significantly enhanced, the highest was about 49 percentage over that of wild type.4. The data of total protein and amino acid analyses revealed that there was no or very little effect of the introduced target genes on total seed protein and total amino acid contents in transgenic rice. However, in transgenic rice with significantly increased free lysine level, some other free amino acids'content were also changed, especially aimino acids related to lysine metabolism pathway, such as threonine and aspartic acid.5. The results from field trials and quality analyses showed that there was no or limited effect of the transgenes on main agronomic traits and grain quality in most of the transgneic rice lines. But, in transgenic rice with significantly increased free lysine level, the grains showed more chalkiness, lower amylose content, softer gel consistency, and little lower starch viscosity when compared with those of wild type.