Functional Analysis Of Citrus Transcription Factors CsMYB77 And CsMYB21 In Regulating Fruit Ripening

At present,one of the urgent problems to be solved in China's citrus industry is the concentrated fruit ripening period.There are few early and late-maturing varieties,and the irrational structure of the ripening period seriously reduces the market competitiveness of citrus in China.How to regulate the ripening period of citrus fruits by molecular means,and to analyze the mechanism of fruit ripening is of great significance to the development of citrus industry.Previously,the transcriptomes of Fengjie navel orange and its late-maturing buds were sequenced,and the transcription factors CsMYB77 and CsMYB21 related to fruit ripening were screened.These two transcription factors were transferred into tomato for functional verification.In this study,based on previous studies,the functional verification of tomato fruits overexpressing CsMYB77 and CsMYB21 in the T1 and T2 generations was performed,and the function of the transcription factor in fruit ripening was determined.The main findings are as follows:1.Phenotypic observation and statistics of T1 generation over-expressing tomato showed that compared with the wild-type and the control,Break time of overexpression fruits is 3-6 days later.All the over-expressing CsMYB77 and CsMYB21 lines were analyzed for the expression of the target genes,and each line selected one line fruit seed for T2 seeding.2.Phenotypic observation and statistics of T2 generation over-expressing tomato.Compared with the control,the number of days of broken of the over-expressing fruit was significantly later,and the whole fruit's broken time was delayed by 3-6 days.Compared with the wild type,the break time of only one over-expressing line MYB77 1-3 was significantly later than that of the wild type.The ripening-related physiological indicators of tomato fruits of T2 generation were tested.Ethylene release rate test results showed that the ethylene release rate of wild-type and control fruits began to increase at 34 DAF and reached the peak of release at 39 DAF.However,the ethylene release rate of the over-expressing lines started to increase at 39 DAF,reaching the peak of release at 42 DAF or 45 DAF.The results of high performance liquid chromatographic detection of lycopene in fruit showed that no lycopene was detected in wild-type,control and over-expressing lines fruit at 26 DAF,and lycopene began to accumulate in wild-type and control fruits at 36 DAF,over-expressing lines began to accumulate lycopene at 39 DAF.The above results showed that the tomato fruits over-expressing CsMYB77 and CsMYB21 were later broken than the wild-type and the control fruits,which delayed the fruit ripening process.3.Expression analysis of mature related genes revealed that the expression levels of ethylene synthesis genes SlACS4 and SlACO1,phytoene synthesis gene SlPSY1,mature regulatory transcription factors SlRIN and SlNOR were increased from 36 DAF in wild-type and control.In the over-expressing lines,the expression of these mature related genes began to increase at 39 DAF,which was consistent with the previous statistical results of fruit coloring and physiological indicators,indicating that tomato fruits over-expressing CsMYB77 and CsMYB21 could delay maturation for about 3 days.