Functional Analysis Of The ZmCYP72A5 Gene In Maize

Cytochrome P450(CYP450)is an ancient superfamily of heme thioprotein,which widely exists in the cells of animals,plants,bacteria and fungi,and is involved in the synthesis and metabolism of various substances in the process of biological growth and development.CYP450 oxidase family plays an important regulatory role in plant resistance to insects and herbicides,but their roles in the regulation of maize development are unclear.In this study,the ZmCYP72A5 gene was edited in maize by gene editing technology to uncover its function in maize growth and development.The main results are as follows:The ZmCYP72A5 gene located on chromosome 3 in maize.Its full length is 1853 bp containing the 1581 bp coding region that encodes 526 amino acids.ZmCYP72A5 protein containes a transmembrane domain and a highly conserved P450 superfamily domain at the N-terminus.The three domains that are the unique functional domains of P450 superfamily,including oxygen binding active region,PERF region and heme binding region,are also found in ZmCYP72A5.The phylogenetic tree analysis show that maize CYP72A5 has high homology to CYP72A5 of monocotyledonous plants,closest to CYP72A15 of sorghum and far from dicotyledonous plants.The result of subcellular localization in maize protoplast showed that the ZmCYP72A5 protein was located on the endoplasmic reticulum.The results of qRT-PCR showed that ZmCYP72A5 was expressed in different tissues and organs,but highly expressed in young roots and leaves,especially in pollinated leaves.In order to investigate the biological function of ZmCYP72A5 gene in maize,a ZmCYP72A5-CRISPR/cas9 vector was constructed and transformed into maize inbred line Q319 by the Agrobacterium-mediated maize genetic transformation experiment.The results showed that the plant height of positive ZmCYP72A5-CRISPR/Cas9 transgeneic lines is shorter about 10 cm than that of the control,and the ear height also decreased accordingly,but the total number of internode is not change.The results of anatomical observation showed that the 9th to 11 th internodes and internode cell length of transgenic plants are significantly shorter than that of control.In additional,the heading time of ZmCYP72A5-CRISPR/Cas9 plants were delayed 2-5 days.Interestingly,the growth period of the transgenic lines were significantly longer than that of the control.The difference of chlorophyll content between transgenic plants and controls began to be significant at 20 days after pollination,and the green-stay time of transgenic plant leaves was longer than that of the control.The results of monitoring showed that the photosynthetic rate of transgenic plant leaves were lower than that of the control.Moreover,the expression of ZmSEE1,a marker gene of leaf senescence,was decreased in the transgenic lines.In order to examine the yield of transgenic plant,the 100-grain-fresh-weight of at different day after pollination the 100-grain-dry-weight of seed after harvest was determined and compared statistically.The results showed that the accumulation of dry matter in the early stage was slower than that in the controls,but due to the extension of its growth period,the final 100-grain-dryweight is not decrease.In conclusion,the ZmCYP72A5 gene may be involved in the regulation of maize development and has high application potential value for the cultivation of high-yield maize by biotechnology.