Cloning And Primary Functional Analysis Of MYB28 And MYB29 Gene In Broccoli

The aliphatic glucosinolates which are a large group of nitrogen-and sulfur-containing secondary merabolites disribute widely in the Cruciferous. Studies have shown that the degradation products of the glucosinolates not only play an important role in plant defense and responsing to biotic or abiotic stresses, but also have physiological functions such as anti-cancer. Currently, identification and characterization of new regulators in the synthesis of glucosinolates is the most advanced approach to exploit the potential of glucosinolates. The mechanism of glucosinolates synthesis have been extensively studied in Arabidoposis thaliana. However, the composition of glucosinolates in Brassica oleracea var. italica is more complicated, and its research of mechanism of glucosinolates is also relatively backward. The primary aim of this study is to discover and characterize novel regulators of glucosinolate biosynthesis in broccoli. The BoMYB28 and BoMYB29 of broccoli are cloned, and the research of BoMYB28 and BoMYB29 is as follows:(1) The BoMYB28-1 (KM262765.1), BoMYB28-2 (KM262766.1), BoMYB29-1 (KM262767.1) and BoMYB29-2 (KM262768.1) transcription factors from broccoli were identified as novel regulatoers of aliphatic glucosinolates by using RT-PCR. Bioinformatics analysis of the cloned transcription factors shows that BoMYB28 and BoMYB29 have high sequence homology with those of other species, and the structural domain of BoMYB28 and BoMYB29 contains obvious two tandem repeats of 51-53 amino acids which regard as R2R3 Myb-type HTH DNA-binding domain.(2) Prediction analysis of subcellular localization suggested that the amino acid residue sequences such as LKKRL/LKKLR and LKKLL, which termed as SV40-type nuclear localization signals were deteted in BoMYB28 and BoMYB29 transcription factors using the prediction software of UniProtKB/Swiss-Prot. The full-length cDNAs were cloned into the vector pEGAD conferring a GFP under control of CaMV35S promoter and the infestation of onion epidermis were conducted by Agrobacterium strain LBA4404 carrying pEGAD-BoMYB28 and pEGAD-BoMYB29. The subcellular localization results clearly indicated that the BoMYB28 and BoMYB29 transcription factors are targeted to nucleus, which supporting the hypothesis of transcriptional factors.(3) Expression pattern analysis revealed that the expression of BoMYB28 and BoMYB29 have significant differences among different tissues. The highest transcript levels of all the cloned transcription factors are found in the flower head of broccoli. However, there are the lowest expression of these in the broccoli seeds. After MeJA and glucose treatment, the transcript level of BoMYB28 and BoMYB29 are increased gradually, and reached their peaks after the treatment of 15min or 2h. Then, the transcript level returned to the original level after 24h.(4) The full-length cDNAs were cloned into the vector pBIl21, and Arabidoposis thaliana plants over-expressing BoMYB28 and BoMYB29 under the control of CaMV35S promoter were generared using A. tumefaciens-mediated transformation. Analysis of metabolic shows that significant change of the content of aliphatic glucosinolates are detected as the increase transcript level of BoMYB28 and BoMYB29 in the transgenic Arabidoposis thaliana. In the BoMYB28 over-expression line, the main short-chain aliphatic glucosinolates such as 3MSOP,4MSOB and 5MSOP, are increased widely. However, the content of long-chain aliphatic glucosinolates like 8MS00 remained unchanged. In the BoMYB29 over-expression line, the content of both short-chain and long-chain aliphatic glucosinolates are inceased remarkably.(5) The special target region of BoMYB28 and BoMYB29 are cloned into the vactor pTCK303. The over-expressing vector and RNAi vector are transformed into the broccoli and verious resistant shoots are generated successfully. However, the root germination of resistant shoots is relatively slow. Presently, the medium of root germination is still in improvement state.