Acquisition And Identification Of Transgenic Maize With Vitamin E And Phytase Traits

Corn is the main feed ingredient for animals,but the content of ?-tocopherol with high-activity in corn seeds is low,and phosphorus in maize seeds mainly exists in the form of phytate,which cannot be effectively absorbed and utilized by monogastric animals.Therefore,synthetic DL-?-tocopheryl acetate and inorganic 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.In this study,two expression vectors with three and four expression cassettes stacked were constructed,and transformed into corn with Agrobacterium infection method,and transgenic lines with high ?-tocopherol content,high phytase activity and glufosinate resistance were obtained.The molecular characteristics,agronomic traits and nutritional components of transgenic lines were analyzed.The main results are as follows:1.Expression vectors pCAMBIA3301 ZTAO and pCAMBIA3301 ZTGHAO containing three and four expression cassettes were constructed.The vector pCAMBIA3301 ZTAO contained a ZmTMT expression cassette,with an embryo-specific promoter 13387 and a Glb1 terminator;an phytase gene AO expression cassette,with LEG1 terminator and endosperm-specific promoter 123387 and Bar expression cassette of glufosinate resistance gene,with CaMV35 S constitutive promoter and 35 S polyA terminator.The vector pCAMBIA3301 ZTGHAO contains the above-mentioned three expression cassettes and the soybean melanoate transferase gene GmHPT expression cassette driven by the embryo-specific promoter 2941 and terminated by the NOS terminator.Agrobacterium infection was used to infect maize immature embryos,and then obtained transgenic maize ZTAO and ZTGHAO.ZTAO transformants had 2 transformation events;ZTGHAO had 4 transformation events.The transgenic plants of ZTAO and ZTGHAO were crossed with the inbred line Zheng 58/Chang 7-2 to obtain F1,then backcrossed for 5 generations,self-pollinated 2 generations to BC5F3.2.Southern blot infers that ZTAO-1# and ZTGHAO-1# has 1 copy;The 5' and 3' flanking sequence of ZTAO-1# and ZTGHAO-1# has been analyzed clearly: The insertion site of ZTAO-1# is > chromosome: AGPv4: 8: 8179701353,which is in the gene gap region.The insertion resulted in the deletion of exogenous and genomic sequences by 93 bp and 131 bp,respectively.The insertion site of ZTGHAO-1# is > chromosome: AGPv4: 7: 6967877,which is in the gene gap region.The insertion resulted in the deletion of exogenous and genomic sequences by 258 bp and 18 bp,respectively;RT-PCR and Western blot analysis showed that the expression levels of ZmTMT,AO,GmHPT,and Bar were significantly higher than those of the control material.3.More than 90% of ?-tocopherol in ZTAO and ZTGHAO transgenic maize is converted into ?-tocopherol,the content of ?-tocopherol in ZTAO-1# and 2# is 51.58 mg/kg and 66.18 mg/kg,respectively,it is 4.9 fold and 6.3 fold of wild type;the content of ?-tocopherol in ZTGHAO-1#,2#,3# and 4# reached 54.91 mg/kg,52.32 mg/kg,44.73 mg/kg and 37.54 mg/kg,were 5.4 fold,5.17 fold,4.42 fold and 3.6 fold of wild type.The total amount of vitamin E in ZTGHAO-1#,2#,3#,4# reached 101.03 mg/kg,96.99 mg/kg,86.24 mg/kg and 66.07 mg/kg,which are 1.37 fold,1.31 fold,1.17 fold and 1.09 fold of wild type.4.The phytase activity in ZTAO-1# and 2# reached 10884.74 U/kg and 12864.53 U/kg,respectively;ZTGHAO-1#,2#,3# and 4 # was 9104.70 U/kg,10412.16 U/kg,14509.70 U/kg and 15549.37 U/kg,respectively.5.In addition,there were no significant differences on agronomic traits(plant height,ear position,rows number of per ear,kernel number of per row,cob diameter and ear length),germination rate and pollen activity between transgenic lines and wild type.And there was no significant difference on nutrient component(18 amino acids,crude fat,crude protein,dry matter,calcium)between ZTAO-1# and wild type,slight difference but without adverse effect between ZTAO-2#,ZTGHAO-1,ZTGHAO-2#,ZTGHAO-3# and wild type.These transgenic maize,which are rich in ?-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.