Cloning Of Chalcone Reductase Gene Gmchr And Research On Agrobacterium-Mediated Soybean Transformation

Soybean [Glycine max (L.) Merr.] originated in China and has been cultivated for more than five thousand years, which is one of the most important oil crops in the world. Isoflavone is an important polyphenolic comound in the plant secondary metabolites. As a phytochrome, isoflavone play an important role in attracting pollinators and seed dispersers. It can also protect plant from Ultraviolet (UV) harm. As an important part of our diet, isoflavone also have many effects on human health, such as anti-oxidant, regulating cardiovascular systems and preventing cancers. Therefore, there is broad interest in the research of isoflavone in recent years.Chalcone reductase (CHR) is an important reductase, which participate in the isoflavone metabolites of the legume. Together with chalcone synthase, CHR catalysis Coumadin-CoA generating6’-deoxy chalcone, which involves in the downstream biochemical reactions. However, because CHR is a specific enzyme in legume, there is little research about CHR. Therefore, cloning and functional analysis of this gene is helpful to understand the metabolic process of isoflavone. In addition, studying the gene function by transgenic plants is one of the most effective approaches. Therefore, in this study, we did the followings:First, full-length cDNA GmCHR was cloned from the soybean cultivars Nannong1138-2by RT-PCR. Sequence analysis showed that, the length of this gene is1031bp. It has3exons and2introns, and is located on chromosome14. It codes a35489.8-Dalton protein with isoelectric point of6.32, which is composed of315-amino acid. GmCHR nucleotide and its protein sequences showed striking similarity to CHR from other leguminous species, such as Lotus japonicus, Glycyrrhiza glabra, Pueraria Montana, Astragalus mongholicus, Medicago truncatula and Medicago sativa. The mRNA amount of GmCHR was highest in leaves, followed by seeds, flowers, stems and roots. Second, we designed a pair of primers that contained attB sites. After BP reaction we got the entry vector pDONR221-GmCHR. Then by doing LR reaction we obtained the destination vector pMDC83-GmCHR. Finally we transformed pMDC83-GmCHR into Agrobacterium EHA105.To improve the soybean transformation protocol in our laboratory, we introduced a previous constructed vector, pMDC83-GmAOS (allene oxide synthase), which had been built by Wu (2008), into7soybean cultivars, by using Agrobacterium-mediated soybean cotyledonary node transformation method. We also introduced pMDC83-GmCHR into soybean cultivars. Finally, we got6To transgenic plants, and obtained T1seeds from3of the To plants. Then we investigated two factors that affect the seed germination and regeneration rate, which are important to optimize the current soybean transformation protocol. We found that:(1) the seed sterilization duration and genotype affected seed germination rate significantly. A negative correlation between the sterilization duration and seed germination rate was observed. Among the five cultivars, the seed germination rates were:Houzimao95%, Dalihuang77%, Jack74%, Nannong88-3164%and Williams58%.(2) when seeds were clean,7-hour sterilization can keep contamination rate less than5%.(3) The regeneration rates for7soybean cultivars showed Dalihuang has the highest regeneration rate of89.51%, follwed by Houzimao> Jack>Changsan>88-31> Williams>Tai7.