关键词: 玉米, 植酸酶, 维生素E, α-生育酚, 草铵膦抗性

Abstract:

【Objective】Corn seeds are the main feed ingredient for poultry and monogastric animals, but the content of α-tocopherol with high-activity in corn seeds is low, and maize is rich in phosphorus mainly existed in the form of phytate, which cannot be effectively utilized by animals. Therefore, synthesized DL-α-tocopheryl acetate and phosphorus or microbial phytase are supplemented in the feed for optimal animal growth. These methods not only greatly increase the feed cost, but also cause the phosphorus pollution due to the undigested phytate. The aim of this study is to acquire maize seeds rich in α-tocopherol and high in phytase activity, and provide resources for forage maize breeding.【Method】Construct an expression vector with three expression cassette, and introduced into maize through agrobacterium infection method, glufosinate was used for screening; The positive transgenic plants were identified by spraying glufosinate and PCR analysis; RT-PCR analysis of target gene expression at the transcription level; Western blot analysis of target gene expression at the translation level; Phytase activity was measured by spectrophotometric method; vitamin E content was determined by HPLC. At the same time, the differences of other nutrients and agronomic traits between transgenic lines and wild types were compared.【Result】Constructed an expression vector with three expression cassette, phyA2 driven by endosperm-specific promoter 123387, ZmTMT driven by embryo-specific promoter 13387, Bar driven by constitutive promoter CaMV35S, and introduced into maize; Transgenic plants were identified by spraying glufosinate and PCR; Two transgenic homozygous maize lines TPB1 and TPB2 with good target traits were obtained after multiple generations of backcross transfer. RT-PCR and Western blot analysis showed that phyA2, ZmTMT and Bar were all highly expressed in transgenic lines. Phytase activity determination showed phytase activity reached to 10 000-13 000 U·kg ^-1, which is able to meet the needs of animal growth and development, vitamin E content measurement showed more than 90% of γ-tocopherol was transformed to α-tocopherol, and α-tocopherol content increased to 50-70 mg·kg ^-1. In addition, there were no significant differences on agronomic traits between transgenic lines and wild type, and there was no significant difference on nutrient component between TPB1 and wild type, slight difference but without adverse effect between TPB2 and wild type; and the transgenic plants were resistant to BASTA.【Conclusion】These transgenic maize, which are rich in α-tocopherol and phytase, and also with herbicide resistance, can be applied for maize breeding to reduce feed cost, improve the utilization rate of phosphorus and reduce environmental pollution.

Key words: maize, phytase, vitamin E, α-tocopherol, glufosinate resistance