Breeding And Evaluation Of Pyramiding Transgenic Rice With High Lysine

Rice is one of the most important crops in the world, as the staple food for half of the world’s population. It is also the most popular crop in China, which accounts for about30%of the total crop area and40%of the total output of grain. With the improvement of the living standard, people concern more about the nutritional quality of rice. The composition of protein and amino acids in rice is the major criteria for evaluation of rice nutrational quality. However, the protein content of rice only accounts for about5～10%of the seed weight. In addition, as the most limiting essential amino acid, lysine only accounts for about3.5～4.0%of the total protein. Therefore, improving the lysine content of rice is one of the major goals of genetic breeders.In the previous studies of our laboratory, we have successfully increased the free lysine content of rice by genetic modification of the expression of several key enzymes in the branch pathways of lysine biosynthesis and degradation. And we also got some multi-transgenic rice through conventional crossing. In the present study, gene expression analysis, lysine content assay, appearance quality analysis and physicochemical quality study were carried out by using the multi-transgenic rice. This study will be helpful for the breeding and utilization of the high-lysine rice in the future. The main results were as followings:1. Selection of homozygous and multi-transgenic rice lines. Through years of efforts, we have selected some homozygous transgenic rice lines, such as GR-65, GR-14,35S-15, etc, which contains different transgene such as the AK, DHPS and LKR-RNAi. In order to further improve the lysine content of transgenic rice, we corssed the homozygous transgenic line35S-15with GR-65and GR-14, respectively. By multiple generations of selection, we obtained multi-transgenic rice35S-15/GR-14and GR-65/35S-15. Besides, we also got homozygous transgenic rice lines RWY3/(35S-15/GR-14) and RWY3/(GR-65/35S-15) by crossing the multi-transgenic rice with R-WY3, which is resistance to stripe virus disease.2. Expression analysis of the target genes in leaves and endosperm of the transgenic rice. The transcription amounts of AK and DHPS are high in the transgenic rice with AK and DHPS overexpressed. The expression of endogenous LKR/SDH gene is notably inhibited in the LKR/SDH RNAi transgenic rice lines..3. Analysis of the lysine content in the mature seeds of transgenic rice. The results showed that the free lysine content in the mature seeds of transgenic rice has increased remarkbly than the control. The free lysine contents in the homozygous transgenic rice lines35S-15/GR-14and35S-15/GR-65have increased about20times. And the total lysine contents also increased. The lysine content in the polymeric transgenic rice35S-15/GR-14has a remarkable increasement, up to20-50times higher than the parent RWY3.4. Study of the physicochemical quality changes of the high lysine transgenic rice. The seeds of the polymeric homozygous transgenic rice showed darken color and increased chalkiness. The protein content of the milled rice has increased significantly. With the increasement of the lysine content, the amylose content in the transgenic rice is decreased. Compared with the untransformed control, the peak viscosity (PKV), hot paste viscosity (HPV), setback (SBV) and final viscosity (CPV) of the transgenic rice with high free lysine content were decreased remarkably. While the breakdown value (BDV), Peak time (PeT) and from the pulp temperature (PaT) had no change.5. Investigation of the agronomic traits of the high lysine transgenic rice in the field.The results showed that the1000kernel weight, main panicle length and plant height of the transgenic rice increased significantly when compared with those of the wild type. The1000kernel weights of the homozygous transgenic lines GR-65and GR-14have increased while that of line35S-15has decreased significantly. In addition, the1000kernel weight of the polymeric homozygous transgenic rice has also decreased while the available tiller numbers and seed setting rate have no change when compared with those of the untransformed control.