The Study On The Molecular Mechanism Of Anthocyanin Synthesis And Accumulation In Blueberry Fruit Based On The Regulation Of ABA

Blueberry is an economic forest fruit of Vaccinium in Ericaceae.It has unique flavor and nutritional and health value,and is deeply loved by consumers all over the world.Blueberry fruit is rich in anthocyanins,which plays an important role in fruit appearance quality and human health care.Therefore,the study on the mechanism of blueberry anthocyanin synthesis has important theoretical value and practical guiding significance.In this paper,the effects of 0,500 and 1000 mg/L ABA(abscisic acid)on fruit quality,physiological and biochemical indexes,fruit color,anthocyanin content and antioxidant capacity were studied.The effects of external application of 1000 mg/L ABA on the metabolites and gene changes of blueberry fruit from late green fruit to mature stage were systematically analyzed by transcriptome and metabolome techniques.Based on the techniques,the influence on anthocyanin synthesis pathway was analyzed,the key transcription factor MYBA was excavated.Its gene cloning and functional verification were carried out,and the mechanism of anthocyanin synthesis regulation in blueberry fruit was discussed.The specific research results are as follows:(1)The fruit of rabbit eye blueberry variety ’Brightwell’ was divided into six periods from late green fruit to maturity(S1-S6).The fruits of late green fruit S1 were treated with ABA at the concentrations of 0,500 and 1000 mg/L.The basic quality indexes and physiological and biochemical indexes of fruits during S1-S6 period were analyzed.The changes of anthocyanin content in fruits during S3-S6 period were determined by p H differential method and liquid chromatography-mass spectrometry.The results showed that external application of ABA could accelerate the color transformation,ripening and softening of blueberry fruits,increase the accumulation of soluble solids,and improve the content of total anthocyanins and antioxidant capacity of fruits.The effect of 1000 mg/L ABA treatment was better than 500 mg/L;The main anthocyanins in blueberry fruits are petunidin,cyanidin,delphinidin,pelargonidin and peonidin.The treatment with ABA at the concentration of 1000 mg/L can improve their content in varying degrees.(2)The metabolomics of blueberry fruits treated with 0 and 1000 mg/L ABA were analyzed according to S1-S6 period.The results showed that the application of 1000 mg/L ABA significantly affected the overall level of metabolites during S1-S6 period;The content of endogenous ABA increased in S1-S3 period and decreased in S4-S6 period;Among the differential metabolites annotated in lipmaps database,43 differential metabolites are related to flavonoid anabolic pathway;Among the top ten metabolites with significant difference,phenolic acids accounted for 24.1%.By analyzing the metabolite changes in the flavonoid metabolic pathway during S1-S6,it was found that from S3,the external application of 1000mg/L ABA would increase anthocyanin synthesis and reduce the synthesis of other metabolites in the pathway.For mature blueberry fruit(S6 period),when 1000 mg/L ABA was applied to increase anthocyanin content,the content of catechol,phenolic acid,flavonol,tannin and other active substances decreased.(3)The transcriptome of fruits treated with 0 and 1000 mg/L ABA were analyzed according to S1-S6 period,of which the number of differentially expressed genes in S2 period was the largest.KEGG enrichment and annotation results showed that the differentially expressed genes in S5 period were mainly related to flavonoid synthesis pathway.By analyzing the key gene families of ABA signal pathway,it was found that the expression of 7 genes in ABF family was significantly up-regulated in S1-S2 period of ABA treated fruits,and the overall expression of PYL and Sn RK2 family genes did not change and trend significantly.The results of gene expression analysis showed that the structural genes CHS,CHI,DFR and LDOX/ANS related to anthocyanin synthesis in the flavonoid synthesis pathway in blueberry fruits treated with 1000 mg/L ABA were expressed at a low level in S1-S2 and at a high level in S5-S6,which was consistent with the change trend of anthocyanin content in blueberry fruits.Based on Swissprot database annotation and phylogenetic tree analysis,41 potential key genes of anthocyanin biosynthesis were identified,including 33 MYB,4 b HLH and 4 WD40,and according to the relationship between gene expression characteristics and anthocyanin content,MYBA was probably the key transcription factor in anthocyanin biosynthesis control of blueberry fruit.(4)Through the genome sequence alignment analysis of northern highbush blueberry variety ’Draper’,it was found that there were five homologous genes related to anthocyanin synthesis regulation gene Vc MYBA in its genome,and their location and sequence information in the genome were determined.Bioinformatics analysis showed that one copy’s two exons were replaced by transposons and lost its function,and the other four copies were effective copies.Their coding sequences were conservative and the length was 786 bp,but the intron sequences were quite different.According to the sequence characteristics,they are named Vc MYBA1-1,Vc MYBA1-2,Vc MYBA2-1 and Vc MYBA2-2 respectively.The coding sequence similarity between Vc MYBA1-1 and Vc MYBA1-2 is 99.11%,and the introns contained in Vc MYBA2-1 and Vc MYBA2-2 are almost the same;The intron sequence of Vc MYBA1-1 is about1000 bp longer than Vc MYBA1-2,and the intron sequence of Vc MYBA1-2 is 1000 bp longer than Vc MYBA2-1 and Vc MYBA2-2;There are not only ABA response elements,but also light response elements in the four Vc MYBA promoters.Temporal and spatial expression analysis also shows that the expression of Vc MYBA is positively correlated with the anthocyanin content of blueberry fruit under different light quality treatments.It is speculated that Vc MYBA is involved in the regulation of anthocyanin synthesis in blueberry fruit in ABA pathway and light response pathway.(5)According to the Vc MYBA gene sequence of northern highbush blueberry,primers were designed.Four Va MYBA genes were cloned from rabbit eye blueberry variety ’Brightwell’.The coding sequences of Va MYBAs and Vc MYBAs were basically the same,and the similarity was very high.The analysis of temporal and spatial expression characteristics showed that the expression of Va MYBAs could be detected in blueberry fruits in S3 period,increased rapidly in S4 period,and then increased continuously in S5 and S6 periods.The change of expression was positively correlated with the change of anthocyanin content.Ca MV35s-p CAMBIA1301 vector was constructed,and the function of Va MYBAs gene was verified by heterologous expression by transforming tobacco leaves with Agrobacterium tumefaciens.The results showed that the heterologous expression of Va MYBAs gene in tobacco could not induce anthocyanin synthesis.VIGS technology was used to instantaneously interfere with Va MYBAs in ’Brightwell’ fruit and Vc MYBAs transcript in ’Legacy’ fruit.The results showed that the transient silencing of MYBAs transcripts in blueberry fruit resulted in the reduction of anthocyanin components such as petunidin,cyanidin,delphinidin,pelargonidin and peonidin in different degrees.Therefore,MYB transcription factor gene MYBAs plays an important regulatory role in the synthesis and accumulation of anthocyanins in blueberry fruits.(6)The sequence comparison,phylogenetic tree and DNA polymorphism analysis of MYBA gene of 22 varieties covering various classification types of Vaccinium were carried out.The results showed that the sequence difference of MYBA gene in Vaccinium was small,the genetic distance between species was close,and the DNA polymorphism was small.It was considered that the MYBA coding sequence was relatively conservative among Vaccinium species,and the sequence difference was not the main reason for the difference of anthocyanin content in fruits of various varieties of Vaccinium.The MYBA gene sequences of 16 blueberry varieties were obtained by PCR amplification and analyzed with the published genome information of Vaccinium.The results showed that there were 5 copies in the MYBA locus of Vaccinium,but the number of effective copies of MYBA gene was different among varieties and varieties,with a range of 3-5;There are a large number of retrotransposons in MYBA locus,and retrotransposons are related to gene tandem replication events.It is speculated that gene tandem replication events may be the reason for the difference of MYBA loci in Vaccinium.The correlation analysis between fruit anthocyanin content and MYBA effective copy number showed that there was a positive correlation between MYBA effective copy number and fruit anthocyanin content;Through transcriptome analysis,it was found that the effective copy number of MYBA gene was positively correlated with the expression levels of CHI,F3 H,DFR,ANS and UFGT genes.It is speculated that the effective copy number of MYBA gene is an important factor in regulating the synthesis and accumulation of anthocyanins in Vaccinium fruits.