基于转录组测序的平欧杂种榛S位点相关基因的克隆与表达分析

发布时间：2018-08-10 18:08

【摘要】：平欧杂种榛(Corylus heterophylla Fisch.×Corylus avellana L.)为中国榛属植物(Corylus)的主要栽培品种,是重要的经济林树种,其多表现为自交不亲和。平欧杂种榛的栽培需要配置合理的优良授粉品种,同时其种质创新目前也主要通过杂交育种获得,因此,平欧杂种榛自交不亲和性的研究对其丰产栽培和种质创新均具有极重要的意义。经典遗传学认为榛属植物属于孢子体型自交不亲和,但对其S位点及其相关基因的分子研究还处于起步阶段。本研究以平欧杂种榛的主要栽培品种‘达维’为材料,对其不同亲和性授粉、授粉后不同时间的雌蕊进行了转录组测序分析,并基于其测序数据筛选和验证了适合榛属植物实时荧光定量PCR的稳定内参基因;在此基础上,利用RACE技术基于转录组测序数据对平欧杂种榛S位点相关基因进行了克隆,以期从分子层面对榛属自交不亲和性进行深入研究。主要获得以下结果:1.平欧杂种榛转录组测序本研究利用Illumina HiSeq 2000平台对平欧杂种榛不同亲和性组合、授粉后不同时期的雌蕊进行转录组测序,总计产出42,046,035,660 nt数据,注释Unigene 56,177个,比对和预测编码蛋白框53,054个,涉及COG分类26个、GO分类55个,注释代谢通路128个。IC4处理与其他处理的样品比对结果表明,在自交不亲和组合中,授粉后4 h是榛子自交不亲和研究的关键处理和时期。此外,基于转录组测序数据的Blast比对和SMART比对结果证实,榛属植物确实属于孢子体自交不亲和。2.平欧杂种榛实时荧光定量PCR内参基因的筛选与确定基于转录组测序数据和前人的相关研究共筛选获得12个候选内参基因。反转录PCR产物琼脂糖凝胶电泳和实时荧光定量PCR溶解曲线均表明,针对候选内参基因所设计的引物特异性均良好。引物评价结果显示,引物扩增效率E值在86.3-121.6%之间,R2值均大于0.98,其线性回归方程可信。通过程序geNorm、Normfider、Bestkeeper和Delta Ct法基于荧光定量PCR所获得的Cq值,分别评估了候选内参基因在24个样品(不同组织器官、自交授粉后不同时间的雌蕊与不同亲和性授粉后4h的雌蕊)中的稳定性。最终筛选出本试验样品最合适的稳定内参基因ChaActin和ChaEF1-α,并利用已发表的欧洲榛Cav Prx基因对其进行了表达分析的验证,结果表明上述内参基因在榛属植物基因表达分析中具有实用性,可用于其自交不亲和相关基因的表达分析。3.平欧杂种榛S位点相关基因的克隆与分析基于筛选所得的Unigene,利用RACE克隆技术,克隆获得了8个S位点相关基因(ChaTHL1/ChaTHL2,ChaMLPK,ChaExo70A1,ChaARC1,ChaMOD1/ChaMOD2和ChaKAPP)的cDNA编码区全长序列。进行了编码框预测、蛋白质理化性质与结构分析、氨基酸序列结构域和功能预测及进化分析等生物信息学分析,结果表明:1)ChaTHL1和ChaTHL2所编码的蛋白均为稳定性蛋白、其存在一个thioredoxin结构域、具有蛋白质二硫氧化还原酶活性,在进化中属于THL类蛋白;2)ChaMLPK为碱性亲水性稳定蛋白、富含磷酸化位点(53个)、具有一个STKs结构域,与核桃(Juglans regia)的蛋白激酶APK 1A序列亲缘关系最近;3)ChaARC1为酸性亲水性稳定蛋白,具有泛素化转移酶活性、存在一个U-box和两个ARM结构域,与核桃含有U-box结构域的蛋白序列亲缘关系最近;4)ChaExo70A1为碱性亲水蛋白、存在一个Exo结构域、属于胞外分泌复合体,与核桃Exo70A1亲缘关系最近;5)Cha MOD1和ChaMOD2均为碱性疏水蛋白,属于PIP家族,是膜的组成部分、参与物质转运过程;6)ChaKAPP为酸性亲水蛋白、具有磷蛋白磷酸酶活性、存在一个FHA和一个PP2C结构域,与核桃PP2C 70序列亲缘关系最近。以本研究筛选所得的稳定基因(ChaActin和ChaEF1-a)作为双内参基因,利用荧光定量PCR对上述基因进行了表达分析,结果表明:1)ChaMLPK和ChaMOD1/ChaMOD2在花粉中几乎无表达,但在其他组织中均有表达,其余5个基因为组成型表达,在各组织器官中均有一定量的表达存在;2)在自交不亲和授粉后24小时内ChaTHL1/ChaTHL2、ChaARC1和ChaKAPP均在2 h时表达量达到最高,而ChaMLPK,ChaMOD1和ChaExo70A1在24 h时达到最高,ChaMOD2则在1 h时达到最高值。
[Abstract]:Corylus heterophylla Fisch. * Corylus avellana L. (Corylus heterophylla L.) is the main cultivated species of Corylus in China. It is an important economic forest species. Most of them are self-incompatible. Therefore, the study on self-incompatibility of Hazelnut hybrids in Pingyou is very important for its high-yield cultivation and Germplasm innovation. Classical genetics considers that Hazelnut belongs to sporophytic self-incompatibility, but the molecular study on S locus and related genes is still in its infancy. The S-locus of Hazelnut hybrid Hazelnut was analyzed by transcriptome sequencing based on different compatibility pollination and different time after pollination, and the stable internal reference genes suitable for real-time fluorescence quantitative PCR of Hazelnut were screened and validated according to their sequencing data. The main results are as follows: 1. Transcriptional sequence of Pingyou hybrid hazelnut. In this study, different compatibility combinations of Pingyou hybrid hazelnut were sequenced by Illumina HiSeq 2000 platform, and the pistils at different stages after pollination were sequenced. 42,046,035,660 NT data, 56,177 Unigene annotations, 53,054 coding frames, 26 COG classifications, 55 GO classifications and 128 annotation metabolic pathways were compared with other treatments. The results showed that 4 h after pollination was the key treatment and time for self-incompatibility study of hazelnut. In addition, the results of Blast and SMART comparisons based on transcriptome sequencing data confirmed that Hazelnut plants were sporophytic self-incompatibility. 2. Screening and identification of internal reference genes in real-time fluorescence quantitative PCR of Pingyou hybrid Hazelnut. Twelve candidate internal reference genes were screened based on transcriptome sequencing data and previous studies. Both agarose gel electrophoresis and real-time fluorescence quantitative PCR showed that the primers designed for the candidate internal reference genes had good specificity. Primer evaluation showed that the amplification efficiency E was between 86.3-121.6% and R2 was above 0.98. The linear regression equation was credible. The program geNorm, Normfider, Bestkeeper and Delta CT were used to determine the primers. The Cq values obtained by fluorescence quantitative PCR were used to evaluate the stability of the candidate endogenous reference genes in 24 samples (different tissues and organs, pistils at different time after self-pollination and pistils at 4 h after different compatibility pollination). Finally, the most suitable stable endogenous reference genes ChaActin and ChaEF1-alpha were screened out and the published Europeans were used. The Cav Prx gene of Corylus heterophylla L. was verified by expression analysis. The results showed that the above-mentioned internal reference genes were useful in gene expression analysis of Corylus L. and could be used for expression analysis of self-incompatibility related genes. 3. Cloning and analysis of S locus related genes of Pingyou hybrid Corylus heterophylla L. Based on the screened Unigene, RACE cloning technology was used. The full-length sequences of eight S-locus-related genes (ChaTHL1/ChaTHL2, ChaMLPK, ChaExo70A1, ChaARC1, ChaMOD1/ChaMOD2 and ChaKAPP) were cloned and cloned. The proteins encoded by ChaTHL1 and ChaTHL2 are all stable proteins, which have a thioredoxin domain with disulfide reductase activity and belong to THL-like proteins in evolution; 2) ChaMLPK is an alkaline hydrophilic stable protein, rich in phosphorylation sites (53), with a STKs domain, and a protein kinase APK of Juglans regia. ChaARC1 is an acidic hydrophilic stabilizing protein with ubiquitination transferase activity, and has one U-box and two ARM domains, which are most closely related to the protein sequence containing U-box domains in walnut; 4) ChaExo70A1 is an alkaline hydrophilic protein with an Exo domain, belonging to the exocrine complex of walnut Exo. Cha MOD1 and Cha MOD2 are both alkaline hydrophobic proteins, belonging to the PIP family, which are part of the membrane and participate in the transport of substances; 6) ChaKAPP is an acidic hydrophilic protein with Phosphoprotein Phosphatase activity, and has a FHA and a PP2C domain, which are closely related to the PP2C 70 sequence of walnut. Stable genes (ChaActin and ChaEF1-a) were used as double internal reference genes. The results showed that: 1) ChaMLPK and ChaMOD1/ChaMOD2 were almost not expressed in pollen, but they were expressed in other tissues. The other five genes were constitutively expressed and expressed in various tissues and organs. The expression of ChaTHL1/ChaTHL2, ChaARC1 and ChaKAPP reached the highest level at 2 h, ChaMLPK, ChaMOD 1 and ChaExo70A1 reached the highest level at 24 h and ChaMOD2 reached the highest level at 1 h after self-incompatibility pollination.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S664.4

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