【作者】 胡雪；

【导师】 任喜峰；

【作者基本信息】 华中农业大学 ， 农业硕士（专业学位）， 2023， 硕士

【摘要】 MicroRNA是一类普遍存在于真核生物中的非编码的内源小分子RNA,主要在转录后水平对基因的表达起负调控作用。研究表明miRNA广泛参与植物的生长发育过程,其中一些特定的miRNA参与植物的花药发育,是导致花药发育异常,雄性不育的重要原因。目前,对于miRNA在花药发育方面的研究大部分集中在水稻、棉花、油菜等作物上,而小麦是基因组复杂的异源六倍体,其miRNA调控不育机理相关研究报道较少。因此,研究调控小麦雄性不育的miRNA,对解析小麦雄性不育的机理具有重要的意义。本研究以小麦温敏雄性不育系BNS366在低温不育条件和正常可育条件下的花药为材料,通过Small RNA测序技术鉴定到一些小麦BNS366花药在不同育性中差异表达显著的miRNAs,并且利用生物信息学手段对差异表达的miRNAs以及相应的靶基因进行筛选和初步的功能分析,主要研究结果如下:1.小麦不育系BNS366不同播期的育性表现:分别在10月、11月、12月和1月中旬对小麦BNS366进行大田播种,4个播期的结实率分别为14.76%、28.83%、42.5%和84.1%。同时,对不同播期小麦BNS366的花粉进行细胞学观察,其中第I期,花粉粒干瘪,且I2-KI染色呈黄褐色,绝大部分表现不育;到第Ⅳ期时,绝大部分花粉被染成蓝黑色,花粉表现可育。2.小麦BNS366不同育性花药的Small RNA测序:取不同时期不育和可育花粉构建NN1(可育花粉母细胞时期)、NN2(可育四分体时期)、SL1(不育花粉母细胞时期)和SL2(不育四分体时期)4个文库,测序共获得54,318,314个raw reads,过滤后,SL1,SL2,NN1 和 NN2 文库分别有 11,858,471,13,455,460,13,823,035 和14,434,405个cleanreads。经比对共鉴定到929个miRNAs,包括81个已知miRNAs和 848 个新 miRNAs。3.差异表达miRNAs筛选:通过表达量的显著性分析,从81个已知miRNAs中筛选到21个差异表达的miRNAs,另外从848个新的miRNAs中选择到8个差异表达倍数最大的新miRNAs,共得到29个差异表达的miRNAs。通过对这29个差异表达的miRNAs靶基因进行预测,共得到1068个靶基因,其中有25个靶基因在小麦穗部高表达或特异表达,它们主要受到13个miRNAs的调控。4.qPCR对miRNA和靶基因分析:利用实时荧光定量PCR分析发现miR9662a、miR5062、miR9662b、miR9653a 和 miR9672b 这 5 个 miRNAs 与其潜在调控的靶基因 TraesCS6B02G028000、TraesCS5D02G192700、TraesCS6B02G043100、TraesCS3B02G251700和TraesCS7D02G383200在小麦 BNS366不同育性花药中表现为相反的表达模式,其中,TraesCS3B02G251 700和TraesCS7D02 G383200在穗生长早期阶段、花发育和减数分裂时期维持较高的表达水平,推测其可能在小麦BNS366的育性转换过程中发挥重要作用。5.miRNA的功能分析:分别对7个已报道与育性相关的miRNAs(miR395b、miR5062、miR9672b、miR9676、miR531、miR9653a、miR9670)进行启动子克隆和拟南芥遗传转化。通过对启动子元件预测,发现这些基因的启动子中大多含有低温响应元件LTR或脱落酸响应元件ABRE。同时,对7个miRNAs在拟南芥中进行转化,完成了阳性转化材料筛选,后续通过对这些miRNAs组织表达模式分析来解析BNS366的育性调控机制。综上,本研究通过Small RNA测序技术对温敏雄性不育小麦BNS366在不育和可育条件下的花药miRNA表达谱进行分析,发现了一些可能参与小麦BNS366育性调控的miRNAs,并挖掘了参与BNS366育性转换的潜在靶基因,为后续解析miRNA对温敏雄性不育小麦BNS366的不育机制提供参考。更多还原

【Abstract】 MicroRNAs are a class of non-coding endogenous small RNA molecules and widespread around eukaryotes,which mainly play a negative regulatory role in gene expression at the post-transcriptional level.Many studies have shown that miRNAs are widely involved in the growth and development of plants.Some specific miRNAs are involved in the anther development of plants,which is an important cause of abnormal anther development and male sterility.At present,most of the studies on miRNA in anther development are focused on rice,cotton,rape and other crops.There are few reports on the mechanism of miRNA regulation of infertility in wheat which is a heterohexaploid plant with the complex genome.Therefore,it is of great significance to study the miRNA regulating wheat male sterility for analyzing the mechanism of wheat male sterility.In this study,the anthers of wheat thermo-sensitive male sterile line BNS366 under low temperature sterile conditions and normal fertile conditions were used as materials.The miRNAs expressed during anther development of wheat BNS366 were identified by Small RNA sequencing technology,and the significant differential expression miRNAs and their target genes were screened and analyzed by bioinformatics methods.The main results are as follows:1.The fertility performance of wheat sterile line BNS366 at different sowing dates:BNS366 was sown in the field in October,November,December and mid-January,respectively.The seed setting rates of the four sowing dates were 14.76%,28.83%,42.5%and 84.1%,respectively.At the same time,the pollen of wheat BNS366 in different sowing dates was observed by cytology.In the first sowing date,the pollen grains were shriveled,and the I2-KI staining was yellowish brown,and most of them were sterile.At the IV sowing date,most of the pollen was stained blue-black,and the pollen was fertile.2.Small RNA sequencing of different fertility anthers of wheat BNS366:Four libraries of NN1(fertile pollen mother cell stage),NN2(fertile tetrad stage),SL1(sterile pollen mother cell stage)and SL2(sterile tetrad stage)were constructed from sterile and fertile pollen at different stages.A total of 54,318,314 raw reads were obtained by Small RNA sequencing.After filtration,the libraries of SL1,SL2,NN1 and NN2 had 11,858,471,13,455,460,13,823,035 and 14,434,405 clean reads,respectively.A total of 929 miRNAs were identified,including 81 known miRNAs and 848 new miRNAs.3.Screening of differentially expressed miRNAs:through the significant analysis of expression levels,21 differentially expressed miRNAs were screened from 81 known miRNAs,and 8 new miRNAs with the largest differential expression folds were selected from 848 new miRNAs.A total of 1068 target genes were predicted as the target genes for these 29 differentially expressed miRNAs,of which 25 target genes regulated by 13 miRNAs were highly expressed or specifically expressed in wheat spikes.4.qPCR analysis of miRNA and target genes:Real-time quantitative PCR analysis showed that five miRNAs,miR9662a,miR5062,miR9662b,miR9653a and miR9672b,and their potential target genes TraesCS6B02G028000,TraesCS5D02G192700,TraesCS6B02G043100,TraesCS3B02G251700 and TraesCS7D02G383200 showed opposite expression patterns in different fertility of BNS366 anthers.Among them,TraesCS3B02G251700 and TraesCS7D02G383200 maintained high expression levels during early panicle,flower development and meiosis,suggesting that they may play an important role in the fertility transformation of wheat BNS366.5.Functional analysis of miRNA:Promoter cloning and Arabidopsis genetic transformation of seven reported fertility-related miRNAs(miR395b,miR5062,miR9672b,miR9676,miR531,miR9653a,miR9670)were performed.By predicting the promoter elements,it was found that most of the promoters of these genes contained the low temperature response element LTR or the abscisic acid response element ABRE.At the same time,seven miRNAs were transformed into Arabidopsis thaliana,and the positive transformation materials were screened.Subsequently,the fertility regulation mechanism of BNS366 was analyzed by identifying the tissue expression patterns of these miRNAs.In summary,the expression profiles of anther miRNAs in thermo-sensitive male sterile wheat BNS366 under sterile and fertile conditions were analyzed by Small RNA sequencing technology in this study.Some miRNAs and their target genes that may be involved in the regulation for the fertility conversion of BNS366 fertility were found in the anther development,which provided a reference for the subsequent analysis of miRNA on the infertility mechanism of thermo-sensitive male sterile wheat BNS366.更多还原