【作者】 李斌；

【导师】 易干军； 彭新湘；

【作者基本信息】 华南农业大学 ， 植物学， 2021， 博士

【摘要】 香蕉是典型的呼吸跃变型果实,采收后的果实在常温下能很快出现乙烯高峰,导致果实在一周内迅速腐烂变质。短暂的储藏期给香蕉采后的贮运与保鲜带来严峻考验,也极大地限制了香蕉产业的发展。乙烯在香蕉果实成熟过程中起关键作用,但对其具体分子调控网络还不是很明确。本论文中,我们对香蕉基因组中的MaNAC基因进行了系统的分析和鉴定,并进一步深入解析了MaNAC009、MaNAC087和MaNAC094在果实乙烯生物合成中的作用。主要结果如下:1、香蕉NAC转录因子的全基因组鉴定与分析。通过生物信息学分析,在香蕉A基因组中共鉴定到181个香蕉NAC基因,命名为MaNAC001-181。它们的蛋白序列长度从119到1031个氨基酸不等,分子量大小在13.45到114.29k Da之间,等电点p I在4.24至11.00范围内。181个香蕉NAC基因分为15个亚组,分布在12条染色体上,其中123个基因是共线基因。保守结构域分析显示,MaNAC基因出现了10种保守结构域,每个基因中随机带有1-3个保守结构域,多数基因带有3个保守结构域。为了预测与香蕉果实成熟相关的NAC基因,以香蕉果实的转录组数据为基础,依据基因的表达量和上调倍数筛选出10个受乙烯利高度诱导表达的NAC基因:MaNAC009/016/033/040/074/083/094/095/129/131,亚细胞定位显示这10个NAC基因都定位在细胞核中。RT-q PCR结果显示,10个NAC基因具有与RNA-Seq数据相同的表达趋势。2、NAC转录因子对乙烯合成关键基因的调控分析。根据组学数据,挑选了11个在果实成熟过程中显著上调表达的MaNAC基因做进一步分析。11个NAC基因为MaNAC009/016/033/040/074/083/087/094/095/129/131。进化树分析表明MaNAC009/033/040/094/129聚类至一个分支,且具有明显的转录激活特性;MaNAC131具有转录激活功能但为独立进化分支;MaNAC016/074/083/087/095具有较强的转录抑制作用,其中MaNAC074与MaNAC083同属一个进化分支,MaNAC087与MaNAC016同属一个进化分支,MaNAC095为独立分支。双荧光素酶报告实验表明,MaNAC009和MaNAC094能显著增强MaACS1启动子活性,而MaNAC087则能显著抑制MaACS1启动子活性;MaNAC009/033/040/094/129/131能显著增强MaACO1启动子活性,MaNA016/074/083/087能显著抑制MaACO1启动子活性。选择对MaACS1和MaACO1启动子活性影响最显著的MaNAC009/087/094作进一步分析。通过双荧光素酶报告实验筛选出MaACS1pro-2、MaACS1pro-6、MaACO1pro-6启动子片段可能是NAC转录因子结合的主要位点;进一步的凝胶阻滞实验表明,MaNAC009能直接结合MaACS1和MaACO1启动子中选定的结合位点,而MaNAC087不能结合MaACS1和MaACO1启动子中选定的结合位点,说明MaNAC087可能是通过间接的方式调控乙烯合成关键基因的表达来参与成熟过程。烟草瞬时表达结果表明,MaNAC009可以通过调控乙烯合成关键基因的表达促进乙烯合成,说明MaNAC009在香蕉果实后熟过程中起着重要的作用。为了从遗传上验证MaNAC009和MaNAC087对果实成熟的影响,我们利用香蕉原生质体瞬时表达体系筛选了MaNAC009和MaNAC087的多个基因组编辑靶点,结果显示MaNAC009靶点3编辑效率最高,MaNAC087的靶点3编辑效率最高,为进一步研究MaNAC009和MaNAC087在果实成熟中的功能奠定了基础。3、NAC转录因子与MADS-box转录因子的互作分析。亚细胞定位结果显示MaMADS1、MaMADS2和MaMADS5都定位在细胞核中,但它们具有不同的表达模式,在果实成熟过程中,MaMADS1在果肉和果皮中都上调表达;MaMASD2呈组成型表达;MaMADS5在果肉中表达量轻微降低,而在果皮中呈现上调表达模式。双荧光素酶实验表明,MaNAC094可以显著调控MaMADS1和MaMADS5的表达,相反,MaMADS5对MaNAC094也具有明显的调控作用,而MaMADS1轻微抑制MaNAC094的表达,而且MaMADS1、MaMADS2的加入能明显减弱MaMADS5对MaNAC094的调控作用。酵母双杂交、非变性Western blot检测结果表明,MaMADS5蛋白可以通过K结构域形成同源二聚体,推测其可能以同源二聚体的形式调控MaNAC094的表达。同时,酵母双杂交、Bi FC和Co-IP实验发现,MaMADS1和MaMADS2能与MaMADS5的K结构域发生相互作用,说明MaMADS1和MaMADS2可能通过与MaMADS5互作的形式参与调控果实后熟过程中的乙烯的合成。以上结果表明,在香蕉果实成熟过程中,MaNAC094通过与MaMADS5的相互作用形成调控回路来影响乙烯的合成,同时,MaMADS1和MaMADS2通过与MaMADS5的互作参与其中。综上所述,本研究基于转录组学以及记忆功能鉴定的方法对NAC基因调控果实成熟的分子机制进行了解析,结果将有助于丰富果实成熟分子调控网络并为精准分子改良提供理论依据。更多还原

【Abstract】 Banana is a typical climacteric fruit,and its postharvest fruit can quickly release a climax amount of ethylene at room temperature,which leads to fruit decay within a week.The short storage life brings severe challenges to the storage,transportation and preservation of bananas,and also greatly restricts the development of the banana industry.Ethylene plays a key role in the ripening process of banana fruit,but the regulation network on ethylene synthesis is unclear.In this dissertation,we applied bioinformatics methods to systematically analyze and identify the MaNAC gene in the banana genome,and further determined the role of MaNAC009,MaNAC087,and MaNAC094 in the ethylene biosynthesis of banana fruit.The main results are as follows:1.Genome-wide identification and analysis of banana NAC transcription factors.Through bioinformatics analysis,a total of 181 banana NAC genes were identified in the banana A genome,named MaNAC001-181.Their protein sequence length range from 119 to1031 amino acids,the molecular weight are between 13.45 and 114.29 k Da,and the isoelectric point p I ranges from 4.24 to 11.00.The banana NAC genes are distributed on 12 chromosomes,of which 123 genes are collinear.These NAC genes are divided into 15 subgroups after the phylogenetic analysis with its Arabidopsis and rice homologs.The conserved domains analysis indicated that there are 10 kinds of conserved domains in the MaNAC gene,and each gene contains 1-3 conserved domains randomly.Most genes have 3conserved domains.To identify the NAC genes involved in banana fruit ripening,based on the transcriptome data of banana fruit,10 NAC genes(MaNAC009/016/033/040/074/083/094/095/129/131)highly induced by ethephon were selected based on the gene expression patterns and up-regulation folds.Subcellular location results showed that these 10 MaNAC genes were all located in the nucleus.RT-q PCR results showed that the expression patterns of 10 selected MaNAC genes were similar to that of the RNA-Seq data.2.Analysis of the regulation of NAC transcription factors on key genes of ethylene synthesis.According to omics data,11 MaNAC genes that were significantly up-regulated during fruit ripening were choosen for further analysis.The 11 NAC genes are MaNAC009/016/033/040/074/083/087/094/095/129/131.Phylogenetic tree analysis shows that MaNAC009/033/040/094/129 cluster into one branch and have obvious transcriptional activation characteristics.MaNAC131 also has transcriptional activation activity while it belongs to an independent branch.MaNAC016/074/083/087/095 have strong transcription repressive activity,among them,MaNAC074 and MaNAC083 belong to the same evolutionary branch,MaNAC087 and MaNAC016 belong to another evolutionary branch,and MaNAC095 is an independent branch.The dual-Luciferase report(LUC)experiment showed that MaNAC009 and MaNAC094 can significantly enhance the activity of the MaACS1 promoter,while MaNAC087 can significantly inhibit the activity of the MaACS1 promoter.MaNAC009/033/040/094/129/131 can significantly improve the activity of the MaACO1 promoter,whereas MaNA016/074/083/087 can significantly suppress the activity of the MaACO1 promoter.We chose MaNAC009/087/094,which exerts the most significant effect on the activities of MaACS1 and MaACO1 promoters,for further analysis.Through the LUC report experiments,the promoter fragments of MaACS1pro-2,MaACS1pro-6,and MaACO1pro-6 were determined as the potential regions including binding sites of the NAC transcription factor.Furthermore,electrophoretic mobility shift assay(EMSA)confirmed that MaNAC009 could directly bind with the selected fragments of MaACS1 and MaACO1 promoters,while MaNAC087 could not,which indicate MaNAC087 might be participated in the fruit ripening process through other indirect ways to regulate the expression of key genes of ethylene synthesis.The tobacco transient expression assay further proved that MaNAC009 can promote ethylene production by regulating the expression of key ethylene synthesis genes,indicating that MaNAC009 plays an important role in the ripening process of banana fruit.To verify the genetic functions of MaNAC009 and MaNAC087 on fruit ripening,we screened multiple genome editing targets of MaNAC009 and MaNAC087 using the banana protoplast transient expression system.The results showed that target 3 of MaNAC009 and MaNAC087 had the highest editing efficiency,which laid the foundation for further study of MaNAC009 and MaNAC087 on banana fruit ripening.3.Interaction analysis of NAC transcription factor and MADS-box transcription factors.The subcellular localization results show that MaMADS1,MaMADS2 and MaMADS5 are all located in the nucleus,but they have different expression patterns.During the fruit ripening process,the expression level of MaMADS1 was up-regulated in both pulp and peel,MaMASD2 was constitutively expressed.The transcription of MaMADS5 was slightly decreased in the pulp,but up-regulated in the peel.LUC assays show that MaNAC094 can significantly regulate the expression of MaMADS1 and MaMADS5.On the contrary,MaMADS5 also has a significant regulatory effect on MaNAC094,while MaMADS1 suppress the expression of MaNAC094.Nevertheless,with the participation of MaMADS1 and MaMADS2,the regulatory activity of MaMADS5 on MaNAC094 was significantly reduced.The experiments of yeast two-hybrid(Y2H),non-denaturing Western blot demonstrated that MaMADS5 protein can form homodimers through its K domain,and implied that the homodimer of MaMADS5 might regulate the expression of MaNAC094.Besides,the analysis of Y2 H,bimolecular fluorescent complementary,and coimmunoprecipitation showed that MaMADS1 and MaMADS2 could interact with MaMADS5 through its K domain.MaMADS1 and MaMADS2 might participate in the regulation of the banana fruit ripening through interacting with the K domain of MaMADS5.All the above results indicate that during the banana fruit ripening process,MaNAC094 and MaMADS5 can form a mutual regulatory loop to affect ethylene synthesis.Simultaneously,MaMADS1 and MaMADS2 might be participated in the regulation of ethylene synthesis by interacting with MaMADS5.In summary,transcriptome and gene function identify were utilized to analyse the molecular mechanism of NAC gene regulation of fruit ripening.Our study will enrich the molecular regulatory network of fruit ripening and provide a theoretical basis for precise molecular improvement.更多还原