Effects Of Different Storage Temperatures On Anthocyanin Synthesis In Red Banana (Musa Acuminata ’red Green’, AAA) And Study Of Function Of MaANS

Red banana(Musa acuminata‘Red Green’,AAA)has red fruit,and the flesh is pink after ripening.There are significant differences in appearance,taste,nutrition and other aspects between Musa acuminata‘ Red Green ’ and ordinary banana.It has high nutritional value and commodity value.The color of Musa acuminata ‘Red Green’ is mainly affected by anthocyanin,and the current research on the synthesis and accumulation of anthocyanin in Musa acuminata ‘Red Green’ is still relatively weak.Therefore,it has important theoretical significance and practical significance to study the mechanism of Musa acuminata ‘Red Green’ anthocyanin synthesis and accumulation.In this study,the effects of different storage temperatures on anthocyanin synthesis in Musa acuminata ‘Red Green’ were studied.Anthocyanin synthase gene(ANS/ LDOX),an important gene in anthocyanin synthesis pathway of Musa acuminata ‘Red Green’ was cloned and analyzed.The main research results are as follows:1.Effects of different storage temperatures on the synthesis of anthocyanins in Musa acuminata ‘Red Green’The effects of different storage temperatures of 10 ℃,19 ℃ and25℃ on the hardness of Musa acuminata ‘Red Green’ fruit,changes in anthocyanin content and gene expression of anthocyanin pathways were studied,and the results showed that the storage period of 10℃ was about twice that of 19℃ and 25℃,and the relatively low temperature delayed its anthocyanin synthesis,but could prolong storage period.When the bananas were stored for 10 days,their commercial appearance value decreased at 19 ℃ and 25 ℃.And expression of the anthocyanin pathway-related genes was analyzed,the results showed that,the expression of CHS,CHI,F3 H,DFR and ANS genes of banana stored at10℃ was higher than that of the control and other temperature treatments.temperature.In summary,10 ℃ is more suitable for the storage temperature of Musa acuminata ‘Red Green’.2.Cloning of and subcellular localization analysis of Ma ANS gene from Musa acuminata ‘Red Green’The full-length sequence of Ma ANS gene was successfully cloned by RT-PCR technology,the CDS region of Ma ANS 1083 bp,encoding 360 amino acids,is a hydrophilic unstable protein,20G-Fe II＿Oxy highly conserved domain with ANS protein.Among promoter cis elements,there are light response elements,abscisic acid response elements,MYB binding site response elements.Ma ANS protein is most closely related to plantain and it also clustered with monocotyledonous plants.Subcellular localization showed that Ma ANS localized in the cytoplasm,which may indicate Ma ANS not only affects anthocyanins synthesis but also maintains cell structure stablity.3.Overexpression of Ma ANS in Arabidopsis thalianaThe p SAK277-Ma ANS plant overexpression vector was constructed and tansformed to Arabidopsis thaliana by floral dipping.The results showed that the stems of transgenic Arabidopsis thaliana showed a pale pink color,and the leaf surface and leaf back base showed purple,and the anthocyanin content of the leaves and stems of the transgenic plants was higher than that of wild-type Arabidopsis thaliana,respectively were about 3.24 times and 3.13 times higher than the control.The difference in flower was not significant;the expression of Ma ANS gene in the stems and leaves of transgenic plants was extremely high,more than 400 times that of wild-type Arabidopsis thaliana.4.Construction of Ma ANS gene PTG-Cas9 silencing vector and its stable transformation in Musa acuminata ‘Red Green’Using the modified CRISPR/Cas9 system,the vector was constructed,the sg RNA expression box containing the target gene was constructed,and the expression box was cut and connected to the expression vector by the ‘ Golden Gate ’ cloning method,and the PTG-Cas9-Ma ANS plant silencing vector was constructed,and transformation of Musa acuminata ‘ Red Green ’ mediated by Agrobacterium was carried out,using thin cross-sections as receptor.The phenotypes of the obtained resistant shoots were completely bleached non-completely bleached shoots.And further sequencing and analysis were carried out using genome-specific primer target site mutations,and the results showed that deletion and replacement of bases occurred at the target sites,resulting in translation errors and loss of their original function.