Functional Identification Of Plum PsMYB-75 And Screening Of Differential Genes Before And After Plum Flower Bud Germination

Plum(Prunus salicina Lindl.)Is a plant of the genus Prunus in the family Rosaceae,and is a deciduous fruit tree widely cultivated in China.Plums originated in the Yangtze River Basin,and it takes 700-1000h(≤7.2 ℃)cooling capacity to complete the normal flower bud differentiation;Guangdong belongs to the south subtropical climate and is the southern marginal cultivation area of plums.It has evolved in Guangdong after long-term natural selection and artificial selection A group of characteristic sub-tropical species with low cold demand is formed.Guangdong plums have a variety of fruits and colors,including ‘Sanhua’ plums with red skin and red meat,’Zao Li’ with red skin and yellow meat,and ’Nan Hua Li’ with green skin and yellow and green meat.Color is an important economic indicator of fruit quality.The formation of fruit color is generally the production of pigments.The synthesis of anthocyanins in the fruit is the main reason for the red color of the fruit.The researchers found that the MYB transcription factor is an effective transcription factor that regulates anthocyanin synthesis.In this experiment,"Conghua Sanhua Li" of red-skinned red meat and "Sanyue Li" of red-skinned yellow meat were used as experimental materials to verify the function of the transcription factor Ps MYB-75 related to anthocyanin synthesis of its fruit;The transcriptome data before and after flower bud germination of Huali and Sanyueli were screened for differential genes.(1)The c DNA of ’Conghua Sanhua Li’ was used as a template to clone Ps MYB-75 and bioinformatics analysis of Ps MYB-75.The analysis of ORF finder,Ex PAsy,Promoter and other software showed that the maximum ORF of Ps MYB-75 was 732 bp,encoding a total of 243 Amino acids,the molecular mass is about 283,87.89,the theoretical isoelectric point is 6.91,the number of atoms is 393.5;the highest amino acid in the protein sequence is Leu,accounting for 9.9%;the lowest amino acid is His 1.6%;protein instability index is 49.17;by predicting the hydrophobicity of its protein,the protein is found to be hydrophilic.A multiple comparison was made between the amino acid sequence of Ps MYB-75 and the protein sequences of the MYB genes of the remaining 13 species to establish a phylogenetic tree,and it was found that it had the closest genetic relationship with apricots.(2)Silencing treatment of Ps MYB-75 in ’Conghua Sanhua Li’ fruit showed that its mature fruit showed defects in coloring,anthocyanin synthesis was blocked,and anthocyanin synthesis related structural genes F3’H,UFGT,ANS/LDOX With Ps MYB-75,the expression level decreased significantly.The results of subcellular localization experiments indicate that Ps MYB-75 is localized in the nucleus.Transcription self-activation experiments confirmed that Ps MYB-75 is a transcriptional activator.Gateway technology was used to construct the Ps MYB-75 overexpression vector,and the leaf of K362 tobacco was transformed by leaf disc transformation.The leaves of the transgenic tobacco seedlings turned red.(3)Using "Conghua Sanhua Li" and "Sanyue Li" germinated flower buds and unsprouted flower buds as materials,RNA-seq technology was used to sequence the flower buds and the sequencing results were analyzed.The results showed that the clean data of the samples processed in each period Reaching 6.33 Gb,the percentage of Q30 bases is 92.94% and above.The clean reads of each sample were sequence aligned with the designated reference genome,and the alignment efficiency varied from 90.91% to95.48%.Analysis of the differential genes and comparison of the differential genes in the KEGG database showed that the significant difference genes in the comparative combination of the sprouting flower bud and the unsprouting flower bud of ’Sanyue Li’were mainly enriched in the endoplasmic reticulum protein processing pathway.Comparing budding flower buds with unburied flower buds of ‘Conghua Sanhua Li’,the genes with significant differences are mainly enriched in biosynthetic pathways,photosynthetic pathways,zeatin biosynthetic pathways,cutin,imine,and wax biosynthetic pathways.The comparison between the budding flower buds of ’Sanyue Li’and the unburied flower buds.The genes with significant differences are mainly enriched in sugar metabolism,plant hormone signaling,phenylpropanoid biosynthesis and photosynthesis.