Effect And Application Of Bagging On Regulating Factors Of Relevant Pigmentation Of Actinidia Arguta

As one of the vital sensory traits fruit,color determines its commercial value.Red-fleshed kiwifruit is loved by consumers because of its bright color,and the red color is due to the accumulation of anthocyanins.Therefore,it is very important to carry out anthocyanin-related research to improve the red character of kiwifruit.The biosynthetic pathway of anthocyanins is very complex and influenced by endogenous genes and the external environment,among which light is considered to be a key factor affecting the formation of fruit color.Therefore,it is particularly important to study the mechanism of anthocyanin synthesis in kiwifruit in response to light.However,current researches of light-regulated anthocyanin synthesis have been extensively concentrated in horticultural crops including apples and pears,rarely in Actinidia arguta.In this study,all-red-fleshed kiwifruit ‘Tianyuanhong'(Actinidia arguta)was selected as experimental materials and the fruits at 30 days after full bloom were performed bagging treatment.A total of eight stages including 30,50,70,80,90,100,110,and 120 days after full bloom were set as sampling time.The same stage of non-bagging treatment fruits was also sampled as control.Based on previous RNA-seq and small RNA-seq data,we preliminarily investigated the mechanism of light-regulated anthocyanin synthesis in Actinidia arguta.The main results are as follows:1.The results of the observation of phenotype,measurements of color ratio and hue angle,and the qualitative and quantitative analysis of five anthocyanin components including cyanidin,delphinidin,cyanidin-3-O-galactoside,delphinidin-3-O-galactoside and cyanidin-3-O-xylo-galactoside in non-bagging and bagging fruits at eight stages showed that the dominant components contribute to the redness of flesh,which are cyanidin-3-O-galactoside and cyanidin-3-O-xylo-galactoside.Besides,bagging treatment could inhibit anthocyanin biosynthesis and accumulation mainly by suppressing synthesis of cyanidin-3-O-galactoside.Therefore,we should strictly shaping and pruning,ensure ventilation and light transmission,and maintain normal coloration of all fruits under the shelf.2.Fourteen transcription factor genes related to flesh coloration were screened from previous transcriptome data of ‘Tianyuanhong' flesh during different fruit developmental stages.The correlation analysis between expression level of fourteen transcription factor genes and content of cyanidin-3-O-galactoside and cyanidin-3-O-xylo-galactoside showed that MYB1 and MYB110 may be involved in the accumulation of anthocyanins in ‘Tianyuanhong',and MYB1 might be a regulatory gene that could response to light.AaMYB1 was cloned from the fruit of ‘Tianyuanhong'(Actinidia arguta),and the coding region was 810 bp in length,encoding 269 amino acids.Through protein sequence alignment with transcription factors associated with anthocyanins reported in other species Actinidia chinensis,we found that AaMYB1 contains a conserved domain of R2R3 and is a typical R2R3-MYB transcription factor.The fusion expression vector 35S::AaMYB1-GFP was constructed,transformed into EHA105,transformed into N.benthamian leaves.The results showed that AaMYB1 was localized in the nucleus and can exert transcriptional activation properties in plant cells.The young leaves of tissue-cultured seedlings of Actinidia arguta ‘F5' were further used as explants.After infection,it was found that the callus overexpressing AaMYB1 had red spots,while the wild type did not.Therefore,AaMYB1 may participate in the regulation of anthocyanin biosynthesis in Actinidia arguta.3.Through small RNA-seq at three key stages of ‘Tianyuanhong' fruit coloration,namely 70 d,100 d and 120 d,7,364,610 Unique sequences were finally obtained,ranging from 20 bp to 24 bp in length,among which 24 bp accounted for the largest proportion.A total of 1,063 miRNAs were obtained through deep analysis,including 482 conserved miRNAs and 581 novel miRNAs.Differential expression analysis was performed on 70 d and 100 d,70 d and 120 d,100 d and 120 d samples,and combined with gene functional annotation,eight miRNAs were finally identified as candidate miRNAs related to anthocyanin synthesis of ‘Tianyuanhong'.The expression of these eight miRNAs in samples by two treatments at three critical periods was analyzed by qRT-PCR.The results showed that miR393,miR828,miR396 and miR156 may positively regulate anthocyanin synthesis,while miR167 and miR858 may negatively regulate anthocyanin synthesis.miR396 and miR156 may be involved in the regulation of anthocyanin biosynthesis in Actinidia arguta as a light-response factor.This provides lights for the follow-up study of the miRNA-mediated light response mechanism of anthocyanin biosynthesis in kiwifruit.