Overexpression Of Modified Rice AK And DHPS Genes And Their Effects On Lysine Accumulation In Rice Endosperm

Roughly one-half of the world’s population depends on rice as their staple food.With the improvement of living standards,more and more people are pay attention to rice quality,rather than yield.Rice have superior protein quality and nutritional value for human and animals,but the lack of lysine in rice seed protein results in its nutrient structure imbalance,nutritional quality decline.Therefore,breeders pay more attention to improving the level of first limiting essential amino acid,lysine,in rice.In higher plants,the lysine biosynthesis come from aspartate-family pathway,there are two key enzymes aspartate kinase(AK)and dihydrodipicolinate synthase(DHPS),which are lysine feedback inhibition;while the lysine-ketoglutarate reductase(LKR)regulates lysine catabolism.In the previous studies of our laboratory,a large number of engineering rice were generated,including overexpressed bacterial lysine feedback-insensitive AK and DHPS gene and suppressed LKR gene(35R and GR).In addition,the modified rice AK and DHPS gene,named as AK2 and DHPS1 genes,were obtained and introduced into rice by combination with inhibited LKR expression(35ADL and 35A2D1L).In this study,transgenic lines were screened and identified,and used for analyzing their gene expression,amino acid content,the physical and chemical quality of rice.The main results were showed as followings.1.After identification by PCR during continuous generations,many homozygous transgenic lines were selected,and which were used for further expression and quality analyses.2.Expression analysis of the target genes were performed in developing seeds of the transgenic rice.The expression of LKR gene have inhibited in the transgenic rice containing the LKR-RNAi structure.Bacterial AK and DHPS expression have increased,while rice AK and DHPS have no changed in 35R and GR transgenic rice compared with wild type.Besides,rice AK and DHPS expression have increased in transgenic rice 35ADL and 35A2D1L with overexpression of AK,DHPS,AK2 and DHPS1.3.The free lysine content and total lysine content were analyzed in mature seeds of different transgenic rice and wild type.The results showed that the free lysine content have improved with the increase of 22-,41-,58-,39-,13-fold in transgenic lines 35ADL-207,35ADL-208,35A2D1L-10,35A2D1L-32,35A2DlL-33compared to WT respectively.Meanwhile,their total lysine content also enhanced in these high free lysine transgenic lines.In addition,the free lysine content has obvious accumulated in transgenic lines AK-52,DHPS-22 and their pyramid lines compared with wild type.4.The rice physical-chemical qualities were compared between high free lysine transgenic lines and their wild typeThe apparent amylose content and gel consistency had changed in rice flour of some transgenic lines.Nevertheless,the RVA profiles of pure starch has no significant difference between in those transgenic lines and wild type,in addition to that of Ri-17/DHPS-22 transgenic rice.5.The results from comparison of the main agronomic traits between transgenic rice and wild type showed that the tilling numbers,grain number per panicle of transgenic lines had no change compared with wild type.The main panicle length,grain size and seed setting rate of some transgenic rice different from their parents,but most of the plant height of the transgenic plants have significantly shorter than in wild type.