Isolating The Differential Genes Related To The Fruit Colour Between Ougan And Green Ougan Based On The Transcriptome

Ougan（Sinocitrus suavissima Tseng）which has long cultural history in Zhejiang province is famous as the high nutritional value.Green Ougan which is a bud mutation of Ougan attracts attentions of many researchers since its stay-green characteristic during the fruit ripening proceed.Although several genes related to carotenoids biosynthesis and ABA metabolism have been isolated,the mechanism of stay-green in Green Ougan still unclear.To comprehensively study the difference of molecular mechanism between Ougan and its bud mutation,the transcriptome had been carried out.We use the transcriptome to isolate the candidate genes related to the stay-green in Green Ougan,and provide basic data for the subsequent analysis of the stay-green mechanism.At the same time,the sequence analysis and partial gene function verification of CsERF13 were carried out.The main results of this study were as follows:1.Changes of the fruit colours between Ougan and Green Ougan showed dramatically different during their developmental stages,especially in the ripening when it turned green to yellow in Ougan,while it still stayed green in Green Ougan fruits.The contents of chlorophyll and carotenoids have been detected between them.Based on the results,the chlorophyll contents showed a quicker reduction in Ougan from 165 DAFB,and it decreased less than 0.01 mg·m L^-1at 210 DAFB.However,a much slower reduction has been found in chlorophyll contents during the whole developmental stages of Green Ougan,and it still maintained nearly 0.05 mg·m L^-1at210 DAFB.Unexpectedly,there were no difference of the carotenoid contents between Ougan and Green Ougan.To study the tasty quality of these fruits,the soluble sugar and organic acid contents were detected.It showed that the sucrose contents in the peel of Ougan were higher than Green Ougan.Among the three main organic acids,only malic acid showed difference in peel and flesh between Ougan and Green Ougan.The contents of malic acid were higher in peel of Green Ougan than Ougan,and in flesh of Ougan than Green Ougan.Taken together,the dramatic difference between Ougan and Green Ougan was the fruit colors.2.To isolate the differentially expressed genes（DEGs）,transcriptome sequencing and weighted correlation network analysis（WGCNA）were carried out.A large number of DEGs related to the plant hormones signal transduction pathway had been isolated between Ougan and Green Ougan at 165 DAFB.Among them,the transcript level of CsEBF1 which is related to ethylene signal transduction pathway showed higher in Green Ougan than Ougan during the whole development.Moreover,it interacts with the downstream gene CsEIN3（3 homologous genes）.It is speculated that CsEBF1 may degrade CsEIN3 through ubiquitination in Green Ougan,leading to its insensitivity to ethylene,resulting in the phenomenon of stay-green.The WGCNA showed that the turquoise module had the most closely correlation with chlorophyll a,chlorophyll b and total chlorophyll content（0.81,0.85,0.83）.A total of 54 DEGs related to chlorophyll content were screened from the turquoise module according to the screening criteria of k ME≥0.9 and gene significance value≥0.75;however,no one was related to chlorophyll degradation.CLH1 which was related to chlorophyll degradation was isolated through analyzing all genes of the turquoise module.The transcript level of Cs CLH1-1 and Cs CLH1-2 in Ougan was 2-fold higher than Green Ougan.The transcript levels of several key genes including EBF1,Cs CLH1-1,Cs CLH1-2,FC2,2-MMP,SAG20,ERF109,b ZIP53 were verified by means of real-time q PCR.Dual luciferase assay confirmed that ERF109 had transcriptional inhibition effect on the promoters of Cs CLH1-1 and Cs CLH1-2.The results further showed that ERF109 might affect the stay-green of Green Ougan through transcriptional inhibition of CLH1.3.CsERF13 is one of the most important transcription factors that regulate the degradation of chlorophyll in citrus.However,its transcription level difference in the developmental stages of wild-type Ougan and Green Ougan is very small,and it is impossible to fully clarify the significant difference in chlorophyll content between the two.Thus,we then analyzed the differences in CsERF13 gene sequence.The results showed that there are two CsERF13 sequence types in different tissues of wild-type Ougan and Green Ougan,which are 660 bp and 675 bp,respectively.Both sequences exist in the peel of wild-type Ougan;while Green Ougan contains only 675bp sequence type.This result is also applicable to other citrus varieties.We analyzed the 12 citrus varieties,there are 4 sequence types of CsERF13 with different lengths,which are 657 bp,660 bp,672 bp and 675 bp.The peel of most citrus varieties（which can normally chlorosis）contains CsERF13 sequence types of the above two lengths.The results of dual luciferase analysis showed that the CsERF13 sequence types with different lengths have different transcriptional regulatory effects on PPH,the sequence type of 675 bp has the weaker transcriptional regulatory effect,indicating that the slowing down of the chlorophyll degradation rate of Green Ougan is probably caused by different lengths of the CsERF13 sequence.These DEGs and two sequence types of CsERF13 provided an important basis for the subsequent research on the stay-green mechanism of Green Ougan.