Cloning and expression analysis of MeANR gene in cassava （Manihot esculenta Crantz）leaves with different colors

【Objective】The purpose of the study was to clone and analyze the expression of anthocyanidin reductase gene（MeANR）in cassava，in order to provide theoretical basis for understanding the molecular mechanism of anthocyanin biosythesis in cassava leaves，improving leave color，and cultivating new varieties.【Method】Cassava with 3 leaf colors：Huanan No. 9（SC9，green leaves），mosaic leaf cassava（yellow and green leaves）and purple leaf cassava（purple leaves）were used as materials，the anthocyanin content and proanthocyanidin content were measured. MeANR gene was cloned and analyzed using bioinformatics. Real-time fluorescence quantitative PCR was used to detect the expression pattern of MeANR gene in different tissues and hormone treatments. Transgenic yeast stress experiments were conducted to verify the expression of MeANR under different hormone treatments.【Result】There were significant difference（P<0.05） between anthocyanin content and proanthocyanid content in leaves of SC9，mosaic leaf cassava and purple leaf cassava. Among them，the anthocyanin content in leaves of purple leaf cassava was the highest，reaching 444.59 ng/g，which was as 24.48 and 28.32 times as that of SC9 and mosaic leaf cassava respectively. A MeANR gene was cloned from cassava leaves with 3 different leaf colors respectively，with a coding region（CDS）of 1041 bp，the sequences were consistent，with 7 bp inconsistent with the published sequences in Phytozome database（accession number：Manes.16G016400）. The MeANR protein consisted of 346 amino acids and was a stable non-secreted hydrophilic protein. Its secondary structure was mainly composed of random coils（42.77%）and α-helices（37.28%），with a small proportion of extended chains and β-turns. Subcellular localization was in the chloroplast. The expression level of MeANR gene was the highest in buds，followed by leaves and flowers，and lowest in stems. MeANR gene exhibited different expression patterns under different hormone treatments. After salicylic acid（SA）treatment，the relative expression level of MeANR showed a trend of first decreasing， then increasing，and then decreasing with the prolongation of treatment time. The relative expression level of MeANR gene at 24 h significantly decreased by 68% compared to 0 h. However，after abscisic acid（ABA）and methyl jasmonate （MeJA）treatments，the relative expression level of MeANR gene showed an upward trend，but reached its peak at 24 h of ABA treatment and 9 h of MeJA treatment. The survival rate of MeANR transgenic yeast strain under SA treatment was lower than that of control（yeast strain transformed with pDR196 empty vector），the growth of MeANR transgenic yeast strain treated with ABA and MeJA was better than the control.【Conclusion】There is a positive correlation between the cassava leaf color and anthocyanin content. MeANR is a negative regulatory gene for anthocyanin accumulation in cassava leaves，with obvious tissue expression specificity，and is inhibited by SA，induced by ABA and MeJA.