Cloning And Function Analysis Of CYP83A1 Gnene From Brassica Oleracea Var. Italica

Brassica oleracea var. Italica also called broccoli, is an important member of Brassicaceae plant, originating in Italy. It is containing high nutrition, such as protein, glucose, axunge, vitamin, and so on. The nutritional ingredient of broccoli hailed as "Crown of Greens" ranks first among its counterparts. Glucosinolate are rich in broccoli, while the highest proportion of aphlic glucosinolate are rich in broccoli flowers. Whereby, sulforaphane is hydrolysis product of it and has antitumor activity. The sulforaphane of broccoli is the most anticancer activity in isothiocyanates derivatives, it has attracted much attention because it is more effection for stomach and breast cancer. Hence, we use genetics and further increase the content of aphlic glucosinolate, and then the content of sulforaphane is improved, thereby obtained the most anticancer and disease resistance of broccoli have important significance in both scientific research and medicine science region.In this paper, we take the broccoli as experimental materials, according to the conclusion is that the early stage of the broccoli transcriptome sequencing results blast with the gene sequences in the database, to design the specific primer. Rt-pcr cloning technology are used to get the broccoli CYP83A1 gene sequence of CDS, named Bo CYP83A1. Through the physical and chemical properties analysis and construction of the evolutionary tree, determine Bo CYP83A1 genetic evolutionary status. In addition, construction of 35S∷Bo CYP83A1 plant expression vector, using PCR and RT-PCR technology to arabidopsis transgenic plants on the level of DNA and RNA detection. Using high performance liquid chromatography(HPLC) method decides the of glucosinolates types and content of 35S∷Bo CYP83A1 turn arabidopsis transgenic plants. Explore Bo CYP83A1 genetic role in broccoli aliphatic synthetic way.The main conclusions of this paper are as follows:1. First isolated the broccoli CYP83A1 sequence of CDSBy RT-PCR, first cloned CYP83A1 sequence of CDS from broccoli leaves in four weeks, named as Bo CYP83A1.2. Bo CYP83A1 bioinformatics analysisThe total length of BoCYP83A1 gene sequence is 1509 bp, totally encoding 502 amino acids(Gene registration number is KM111290). Bo CYP83A1 chemical structure, the formula weight of the protein and IP are C2600H4058N674O736S29、57.47 k D and 7.1 separately, is part of stability hydrophilic protein; tertiary structure prediction is the same as secondary structure result; Bo CYP83A1 is the non-secretory protein, it contains no signal peptide. Bo CYP83A1 protein has spiral structure and trans-membrane structure domain. Sequence analysis illustrates that it has higherhomology with Brassica rapa subsp. chinensis,Brassica rapa subsp. Pekinensis, Brassica napus,Raphanus sativus, Arabidopsis lyrata subsp. Lyrata,Arabidopsis thaliana,Brassica juncea,Brassica nigra, 97%, 98%, 98%, 97%, 92%, 89% and 88%respectively, function in differencies.3. Construction of plant super expression vectorThrough npt II to be selection markers, p CAMBIA330035 Su plasmid to drive expression of glucosinolate CYP83A1 by Ca MV35 S.4. Molecular selection of 35S∷BoCYP83A1 plantsIn the paper, we found 15 resistant arabidopsis plants and obtained 14 positive plants in DNA level, include 4 positive plants by RT-PCR, which showed that Bo CYP83A1 has been integrated into wild type arabidopsis genome.5. Identify glucosinolates of 35S∷Bo CYP83A1 plantsGlucosinolates is tested components and content by UPLC, the results show that the total content of aliphatic glucosinolate is increase in 35S∷Bo CYP83A1 transgenic plants. aliphatic glucosinolate include 3MS0 P,4MS0B,4MTB,5MS0 P and 6MS0 H are incresed; 4MS0 B and 4MTB have extremly rise. Indol glucosinolate include I3 M and 4MOI3 M are decresed, but it is not remarkable. The function of Bo CYP83A1 is control aliphatic glucosinolate in plant.6. Phenotypic analysis of 35S∷Bo CYP83A1 transgenic plantsThe transgenic plants compared with wild type appear early phenomenon,and transgenic plants of flower is ealier than the wild type. The result shows that the content of hormone is increase in transgenic plants.