Cloning And Functional Analysis Of Anthocyanin Synthesis-related Regulatory Genes In Guizimai 1

Anthocyanins mainly accumulate in the seed coat of purple wheat,which plays an important role in the tolerance and quality of wheat to abiotic stress and biological stress.A large number of studies have shown that the accumulation of anthocyanins can improve the resistance of plants to abiotic stress.Many transcription factors are involved in the regulation of anthocyanin synthesis,such as MYB transcription factor,b HLH transcription factor,WD40 transcription factor and b ZIP transcription factor.In addition,a large number of studies have shown that the accumulation of anthocyanin content can improve plant resistance to abiotic stress.In order to understand the effects of MYB transcription factor and ABI5 transcription factor on anthocyanin accumulation in Guizimai 1 seed coat,we were selected to MYB transcription factor and ABI5 transcription factor to study.We selected tow transcription factors with obvious changes(GzMYB-7D1 and GzABI5-3A3)from transcriptome data and real-time fluorescence quantitative date for gene cloning and biological information analysis.Through heterologous expression,we further studied the functions of GzMYB-7D1 and GzABI5-3A3 in tobacco and GzMYB-7D1 in rice.The results are as follows:(1)protein primary structure,secondary structure,phosphorylation sites,and promoter cis elements of GzMYB-7D1 and GzABI5-3A3 were analyzed using biological information analysis.The phylogenetic tree was constructed to find GzMYB-7D1 homology with Ta MYB7D,Ta C1-D1-7DS,Ta Ppm1a,Aet MYB7D-As60 and Aet MYB7D-As77,GzABI5-3A3 homology with Ta ABI5D-SH-31,Ta ABI5D-SH-23 and Ta ABI5D-SW-23.(2)We obtained six tobacco overexpression lines of GzMYB-7D1 by Agrobacterium mediated method.We were found that the chlorophyll content in tobacco leaves was significantly higher than that in wild type,and the CAT activity was significantly higher than that in wild type.the expression of Nt PAL,Nt ANS,Nt CHI,Nt F3H,Nt HEMA and Nt CAT in overexpression lines was significantly higher than that in wild type.Above results show that GzMYB-7D1 participate in the regulation of chlorophyll and anthocyanin synthesis in tobacco.(3)To identify the effects of GzMYB-7D1 on the antioxidant capacity of tobacco,the T₁ generation plants of GzMYB-7D1 tobacco overexpression lines were oxidized.The results showed that the chlorophyll content,CAT activity in the leaves of increased significantly after oxidation treatment,while the POD activity decreased significantly after oxidation treatment.The results showed that the expression levels of GzMYB-7D1 and Nt PAL were significantly up-regulated after oxidation treatment,while the expression levels of Nt ANS,Nt CHI,Nt F3H,Nt HEMA and Nt CHLH did not change significantly,but were significantly higher than those of wild type,and the expression level of catalase related gene Nt CAT was also significantly higher than that of wild type.These results suggest that overexpression of GzMYB-7D1 can improve tobacco tolerance to stress by regulating hydrogen peroxide signal pathway and anthocyanin synthesis pathway.(4)GzMYB-7D1 was transferred into rice Zhonghua 11 by Agrobacterium mediated method,and 18 over expressed lines were identified by real-time fluorescence quantitative identification.moreover,in this study,the homologous gene Os MYB108of GzMYB-7D1 in rice were knocked out.after sequencing the amplified fragments,a total of 5 rice knockout lines were found.The phenotypic observation of GzMYB-7D1overexpression lines and GzMYB-7D1 knockout lines showed that there was obvious anthocyanin accumulation in the grain of overexpression lines,but there was no significant difference in knockout lines.Anthocyanins in overexpression rice glume and endosperm were determined and found that anthocyanins in GzMYB-7D1overexpression rice.Above results show that GzMYB-7D1 can positively regulate anthocyanin accumulation in rice.(5)The GzABI5-3A3 was introduced into the tobacco by Agrobacterium tumefaciens mediated method and verified by GUS staining and PCR amplification.The results showed that 8 strains of GzABI5-3A3 overexpressed tobacco were obtained.The results showed that the anthocyanin content of GzABI5-3A3 overexpressed tobacco was slightly higher than that of wild type.Furthermore Nt CHS expression was significantly higher than that of wild type.Above results show that GzABI5-3A3 is involved in anthocyanin synthesis in tobacco,but the effect is small.(6)To identify the effects of GzABI5-3A3 on tobacco under drought stress,this study treated GzABI5-3A3 overexpressed T₁ generation plants with drought.The results showed that the chlorophyll content in overexpressed tobacco increased significantly after drought stress,while the anthocyanin content increased slightly.Antioxidant enzyme(CAT,SOD and POD)activity increased significantly and was significantly higher than that of wild type.The expression of drought related genes was significantly increased and higher than that of wild type in Nt P5CS,Nt ERD10C and Nt NCED3.Above results show that GzABI5-3A3 overexpression can enhance the drought resistance of tobacco.