The Identification And Regeneration Of High Lysine Transgenic Maize

Maize, the largest crop in China, is not only an important source of food, but also an important feed and industrial raw materials. Statistics showed that more than80percent of maize is mainly used for feed. However, due to the poor nutritional quality of normal maize which lacks of essential amino acids such as lysine and tryptophan which are necessary for human and monogastric animals’ health, additives like lysine and other substances must be supplied into feed. Therefore, breed high lysine content maize has great significance not only in promoting the development of the feed and stock industry, but also can ease the malnutrition problem in part of China.The successful use of the opaque-2gene greatly accelerated the process of quality protein maize breeding, but the characteristics of recessive inheritance and endosperm hardness seriously affected the breeding efficiency. Previous studies already proved that using genetic engineering techniques can increase lysine content by reducing or silencing the expression of maize grain endosperm gliadin gene family. Based on that theory, we built22-KD zein gene interfering vectors of maize, and transferred them into maize immature embryo callus by gene gun bombardment, and achieved the following results:1. Mixed the interfering gene (22-KD) which contains zein gene target sequence with the bar gene at ratio of3:1, and bombarded the embryonic callus of maize H99×Hi ⅡB F1generation by gene gun. the interfering gene vectors were bombarded200times, and got3356immature embryos. Through the bar gene resistance screening,90differentiation seedlings were selected, and60events were positve by PCR test in the TO generation. Because of the poor activities of the transgenic plants, the final harvest transgenic plants were9.2. In transgenic plants of T1generation, totally44plants of22-KD transgenic plants were positive by PCR test.3. The grain lysine contents of22-KD T1transgenic positive lines were tested by using Near Infrared Spectrum System. The result showed that, in positive lines of22-KD, contrast with the control, there were3transgenic plants had lower lysine content (0.286%),4lines had higher but not significantly different lysine content (5.2%-23.4%), and2lines reached the lysine content of the standard of quality protein maize (0.419and0.468), which increased46.5%and63.6%respectively.4. The grain gliadin of22-KD T1transgenic positive lines were detected by SAS-PAGE electrophoresis. The results showed that:the22-KD alfa-gliadin of22-KD positive transgenic grains were significantly reduced compared to non-QPM materials B73, YR21and negative control, which indicated that the22-KD interfering gene suppressed the expression of the22-KD gene familyAlthough RNA interference is a relatively mature modern molecular technique used in genetic breedingand quality innovation, and this study has also been received good progress and results, the experiments still need more studies in future. The reason is because the genetic inheritance was not stable, and the genetic background was complicated.