| Foxtail millet is an important cereal crop in China.The main component of yellow pigment is carotenoid in foxtail millet.The composition of carotenoids and its content affect foxtail millet color.Generally speaking,millets with dark yellow color are always regarded as better quality.Carotenoid cleavage dioxygenase plays a significant role in the degradation pathway of carotenoids in plants.The further studies of the Si CCD genes function could lay a foundation for improving the metabolism of carotenoids and the quality of foxtail millet.In this study,two white varieties(ZSG and NMB)and two yellow varieties(JG21 and CN35)were selected as the experimental materials.The millet color,total carotenoids,carotenoids components and genes expression pattern among different varieties were analyzed at different grain developmental stages.CDS and promoter sequence of the SiCCD1 gene were cloned and compared between four varieties.The prokaryotic expression vector of SiCCD1 gene was constructed and its protein was isolated and purified.In order to know the substrate of SiCCD1 protein,we degrade lutein and zeaxanthin in vitro assay.At the same time,we constructed overexpression vector and screened for transgenic Arabidopsis plants.In this study,we aim to reveal the molecular mechanism underlying the carotenoids degradation in foxtail millet from both physiological and the molecular levels.The main results were as follows:1.The color differences between yellow and white varieties were measured by colorimeter at developmental stages of grains.It was found that there were significant color differences among varieties,and the b* value could be used to characterize the yellowness of foxtail millet.By comparing the difference composition and its content of carotenoids among varieties,it shows that the main component of carotenoids in foxtail millet was lutein,and the content of lutein and zeaxanthin in yellow varieties were significantly higher than that of white millet.Lutein and zeaxanthin were decreasing with the grain maturing in foxtail millet,and especially for the zeaxanthin,which could not be detected in white varieties at the end grain stages.At the same time,the b* value index showed different degrees of positive correlation with the total carotenoids content,lutein and zeaxanthin contents in foxtail millet grains.2.Quantitative real-time PCR was used to analyze SiCCD1 expression pattern at different developmental stages.The results shows that the expression of SiCCD1 gene in white varieties wassignificantly higher than that in yellow varieties,especially in S3 stage which is the key stage for millet color changing,the expression level of SiCCD1 gene in NMB cultivate is 11 times higher than that of CN35.Correlation analysis showed that the relative expression of SiCCD1 gene was significantly negatively correlated with carotenoids and its main component lutein content and negatively correlated with zeaxanthin content in mature stage of foxtail millet.It was proposed that the high expression level of SiCCD1 is one of the main reasons causing the decrease of total carotenoids and components in mature stage of white varieties.3.Cloning and comparison of SiCCD1 gene CDS and promoter sequences of four varieties revealed that the full length was 1638 bp and encoded 545 amino acids.Comparing with the other three varieties,the mutation of 511 th amino acid lead to the changes of tertiary structure of the protein in NMB.There is an enhancer-like element involved in anoxic specific inducibility in the promoter sequence of white varieties.4.The prokaryotic expression vector p MAL-c5x-SiCCD1 was constructed and SiCCD1 soluble protein was induced successfully by optimizing the protein induction conditions.Enzymatic reaction in vitro assay showed that SiCCD1 protein mainly aiming at the lutein degration.At the same time,we constructed overexpression vector PGKPGWG-CCD1 and screened for transgenic Arabidopsis plants. |
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