【作者】 张波；

【导师】 赵政阳；

【作者基本信息】 西北农林科技大学 ， 果树学， 2018， 硕士

【摘要】 花青苷含量决定了苹果果皮颜色,目前关于苹果花青苷的研究主要集中于转录水平,而在转录后水平的研究相对较少,小RNA主要在转录后水平发挥功能从而影响植物的表型。本研究首先从不套袋‘澳洲青苹’绿色果皮和套袋处理后红色果皮小RNA高通量测序结果中找出了差异表达的小RNA:MdTAS4-siR81(-)。并对MdTAS4-siR81(-)在果实着色中的功能进行了研究。WD40,bHLH,MYB复合物可通过调控结构基因影响花青苷的形成,目前MYB和bHLH转录因子家族已经在苹果中被全面鉴定,而WD40蛋白还没有相关报道,本研究对苹果新基因组中WD40基因进行了鉴定,分类,进化分析及表达分析。获得了以下结果:1.小RNA高通量测序结果中,不套袋‘澳洲青苹’绿色果皮中MdTAS4-siR81(-)显著高于套袋‘澳洲青苹’红色果皮。在‘澳洲靑苹’和‘新红星’着色过程中,MdbHLH3与花青苷含量有一个相同的变化趋势,而MdTAS4-siR81(-)与MdbHLH3有一个相反的变化趋势。5’RACE证明在‘澳洲靑苹’和‘新红星’中MdTAS4-siR81(-)均可靶向切割MdbHLH3。苹果果皮中瞬时过表达MdbHLH3能够促进结构基因MdDFR、MdANS、MdUFGT的表达从而促进花青苷的积累,而瞬时过表达MdTAS4-siR81(-)可抑制MdbHLH3的表达进而降低花青苷积累。过表达MdbHLH3可以使拟南芥种子中原花青素含量增加,而MdTAS4-siR81(-)可以靶向MdbHLH3进而抑制拟南芥种子中原花青素积累。同时过表达MdbHLH3可以使拟南芥幼苗中积累花青苷,而MdTAS4-siR81(-)可以靶向MdbHLH3进而抑制拟南芥幼苗中花青素积累。这些结果表明在苹果果实着色过程中,MdTAS4-siR81(-)可以靶向MdbHLH3调控苹果果皮中花青苷的形成。2.对苹果新基因进行全面搜索和鉴定,找到346个WD40基因。并根据它们在染色体上的位置命名为MdWD40-1到MdWD40-346。根据结构域组成,将苹果346个WD40基因分成9个亚家族(A-I)。同时对苹果和拟南芥中的WD40基因进行了系统进化分析,结果显示它们被聚成14个不同的组,内含子外显子结构显示每个组有一个相似的结构。此外,共线性分析表明苹果中WD40大规模扩增是由于苹果近代全基因组复制事件所导致。通过GO注释以及与拟南芥同源比对筛选出了与ABA、干旱、低温相关的19个候选基因,定量结果显示19个候选基因在不同组织器官中有一个差异表达。通过ABA,干旱,低温处理,最终找到8个基因(MdWD40-17,24,70,74,219,256,283,307),它们至少响应ABA、干旱、低温三种逆境中的一种。更多还原

【Abstract】 Anthocyanin content determines the color of apple peels.At present,studies on apple anthocyanin mainly focus on the transcription level,but there are relatively few studies on post-transcriptional levels.Small RNA mainly workson the post-transcriptional level and thus affects plant phenotypes.In this study,MdTAS4-siR81(-),the small RNA with differential expression was firstly found out from the high-throughput sequencing result of small RNA of the unbagged ’German apple’ green peel and bag-removed ‘Granny Smith’ red peel.Moreover,the function of MdTAS4-siR81(-)in fruit coloring was studied.WD40,bHLHand MYB compound can affect the formation of anthocyanin through regulating structural genes.At present,MYB and bHLH family of transcription factors have been fully identified in apples,but there is no related report about WD40 protein.Identification,classification,evolution and expression of WD40 gene in the new genome of apple were analyzed.The following results have been obtained:1.In the high-throughput sequencing results of small RNA,MdTAS4-siR81(-)in the green peel of the unbagged ’German apple’ was significantly higher than that of the bag-removed ‘Granny Smith’.In the coloring process of “Granny Smith” and ‘Starkrimson’,MdbHLH3 had an identical change trend with the anthocyanin content,while MdTAS4-siR81(-)had an opposite change trend with MdbHLH3.5’RACE demonstrated that MdTAS4-siR81(-)could realize target cleavage of MdbHLH3 both in “Granny Smith” and “Starkrimson”.The transient overexpression of MdbHLH3 in apple peels could promotes the expression of the structural genes MdDFR,MdANS and MdUFGT to promote the accumulation of anthocyanins,while transient overexpression of MdTAS4-siR81(-)inhibited accumulation of anthocyanin.Overexpression of MdbHLH3 could increase the proanthocyanidin content in the Arabidopsis seeds,while MdTAS4-siR81(-)could target MdbHLH3 and further inhibit the accumulation of proanthocyanidin in the arabidopsis seeds.At the same time,overexpression of MdbHLH3 could accumulate anthocyanins in the arabidopsis seedlings,while MdTAS4-siR81(-)could target MdbHLH3 and inhibit anthocyanin accumulation in the Arabidopsis seedlings.2.A comprehensive search and identification of new apple genes were carried out to find out 346 WD40 genes.They were named as MdWD40-1 to MdWD40-346 according to their positions on the chromosome.Based on the structural domain composition,the 346 apple WD40 genes were divided into 9 subfamilies(A-I).Meanwhile,phylogenetic analysis of WD40 genes in apples and Arabidopsis was made.The results showed that they were polymerizedto 14 different groups and the intron exon structure showed a similar structure in each group.In addition,the collinearity analysis showed that the large-scale amplification of WD40 in apples was caused by the modern genome-wide replication events of Apple.19 candidate genes related to drought and low temperature were selected through GO annotation and homologous comparison with Arabidopsis,and the quantitative results showed that there was one differential expression for the 19 candidate genes in different tissues and organs.Through ABA,drought and cold treatment,eight genes(MdWD40-17,24,70,74,219,256,283and307)were finally discovered,which responded to at least one of ABA,drought,and low temperature.更多还原