| Citrus in our country are mainly mid-varieties and the structure of the species is imbalance seriously.Therefore,it is the key to develop the citrus industry that adjusting the variety structure and expanding the fruit supply period.Previously,the research about citrus ripening related to the metabolism of carbohydrate and carrot has been more mature,but the study of mature related genes is still less.The study on the expression and regulation of ripening genes in citrus fruit is benefical to further reveal its mature molecular mechanism,which is great significance to broaden the ripening period of citrus fruit.In this study,'Fengjie 72-1'(WT)and the late-maturing mutant of 'Fengwan' are as the materials to study Cs MYB21 and Cs MYB77,two fruit ripening related genes.The function of Cs MYB21 and Cs MYB77 was determined preliminarily by transforing Mic-Tom tomato.The main findings are as follow:1.Gene cloning and bioinformatics analysis: The length of gene and promoter region were cloned respectively in WT and MT.And the cis-acting elements were analyzed combined with promoter region.The phylogenetic tree analysis of two gene amino acid sequences with MYB famiy of Arabidopsis thaliana and tomato,using Clustalx 1.81 and Mega 5.1 software,showed that the genetic relationship of Cs2g27940 with AT3g27810 was the closest,Cs3g23950 with AT3g50060 was the closest,so named Cs2g27940 and Cs3g23950 for Cs MYB21 and Cs MYB77 respectively.Futhermore,the primary structures of the two transcription factors,such as protein size,isoelectric point and conserved domain,were predicted.Moreover the results revealed that there was no mutation in the WT and MT of the two full-length genes and the promoter region,and the cis-binding elements of the promoter contained a large number of elements related to the photoreaction and the plant hormone response,indicating that two transcription factors may involve in optical signal or hormone signaling to affect fruit maturation.2.Transcription activity detection of transcription factor: CDS region of two transcription factors were cloned and then we constructed subcellular localization and transcriptional activation vectors respectively.The results showed that two transcription factors were localized in the nucleus by injecting tobacco epidermal cells,which meaned they were nucleus proteins.The fusion expression vector p GBKT7-Cs MYB21 and p GBKT7-Cs MYB77 were transformed into yeast strain AH109,and then cultured on the uncomplete medium.The results showed that Cs MYB21 and Cs MYB77 are typical transcription factors of MYB family.3.Expression pattern analysis: Real-time quantitative PCR was used to determine the expression of two genes in different developmental stages and different tissues.The results showed that Cs MYB77 was mainly expressed in the early stage of fruit development,and Cs MYB21 was mainly expressed in the late stage of development.The expression of two gene was different in different tissues of WT and MT.The results showed that the two genes may be related to fruit ripening.4.Transcription factor function verification: over-expression vector of two target genes were constructed and were in introduced into tomato explant using agrobacterium transformation.We obtained six 35S-MYB21 and 35S-MYB77 transgenic plant respectively by positive identification.The results showed that the overexpression of Cs MYB77 gene can delay the time to brea ker from fruit setting.Compared with the wild type,transgenic plants of Cs MYB21 had a lower germination rate and higher flower dropping.The results revealed that Cs MYB77 may negatively regulate fruit ripening and Cs MYB21 may involved in regulation of the development of the flower. |
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