【摘要】：平歐雜種榛(Corylus heterophylla Fisch.×Corylus avellana L.)為中國榛屬植物(Corylus)的主要栽培品種,是重要的經(jīng)濟林樹種,其多表現(xiàn)為自交不親和。平歐雜種榛的栽培需要配置合理的優(yōu)良授粉品種,同時其種質(zhì)創(chuàng)新目前也主要通過雜交育種獲得,因此,平歐雜種榛自交不親和性的研究對其豐產(chǎn)栽培和種質(zhì)創(chuàng)新均具有極重要的意義。經(jīng)典遺傳學(xué)認為榛屬植物屬于孢子體型自交不親和,但對其S位點及其相關(guān)基因的分子研究還處于起步階段。本研究以平歐雜種榛的主要栽培品種‘達維’為材料,對其不同親和性授粉、授粉后不同時間的雌蕊進行了轉(zhuǎn)錄組測序分析,并基于其測序數(shù)據(jù)篩選和驗證了適合榛屬植物實時熒光定量PCR的穩(wěn)定內(nèi)參基因;在此基礎(chǔ)上,利用RACE技術(shù)基于轉(zhuǎn)錄組測序數(shù)據(jù)對平歐雜種榛S位點相關(guān)基因進行了克隆,以期從分子層面對榛屬自交不親和性進行深入研究。主要獲得以下結(jié)果:1.平歐雜種榛轉(zhuǎn)錄組測序本研究利用Illumina HiSeq 2000平臺對平歐雜種榛不同親和性組合、授粉后不同時期的雌蕊進行轉(zhuǎn)錄組測序,總計產(chǎn)出42,046,035,660 nt數(shù)據(jù),注釋Unigene 56,177個,比對和預(yù)測編碼蛋白框53,054個,涉及COG分類26個、GO分類55個,注釋代謝通路128個。IC4處理與其他處理的樣品比對結(jié)果表明,在自交不親和組合中,授粉后4 h是榛子自交不親和研究的關(guān)鍵處理和時期。此外,基于轉(zhuǎn)錄組測序數(shù)據(jù)的Blast比對和SMART比對結(jié)果證實,榛屬植物確實屬于孢子體自交不親和。2.平歐雜種榛實時熒光定量PCR內(nèi)參基因的篩選與確定基于轉(zhuǎn)錄組測序數(shù)據(jù)和前人的相關(guān)研究共篩選獲得12個候選內(nèi)參基因。反轉(zhuǎn)錄PCR產(chǎn)物瓊脂糖凝膠電泳和實時熒光定量PCR溶解曲線均表明,針對候選內(nèi)參基因所設(shè)計的引物特異性均良好。引物評價結(jié)果顯示,引物擴增效率E值在86.3-121.6%之間,R2值均大于0.98,其線性回歸方程可信。通過程序geNorm、Normfider、Bestkeeper和Delta Ct法基于熒光定量PCR所獲得的Cq值,分別評估了候選內(nèi)參基因在24個樣品(不同組織器官、自交授粉后不同時間的雌蕊與不同親和性授粉后4h的雌蕊)中的穩(wěn)定性。最終篩選出本試驗樣品最合適的穩(wěn)定內(nèi)參基因ChaActin和ChaEF1-α,并利用已發(fā)表的歐洲榛Cav Prx基因?qū)ζ溥M行了表達分析的驗證,結(jié)果表明上述內(nèi)參基因在榛屬植物基因表達分析中具有實用性,可用于其自交不親和相關(guān)基因的表達分析。3.平歐雜種榛S位點相關(guān)基因的克隆與分析基于篩選所得的Unigene,利用RACE克隆技術(shù),克隆獲得了8個S位點相關(guān)基因(ChaTHL1/ChaTHL2,ChaMLPK,ChaExo70A1,ChaARC1,ChaMOD1/ChaMOD2和ChaKAPP)的cDNA編碼區(qū)全長序列。進行了編碼框預(yù)測、蛋白質(zhì)理化性質(zhì)與結(jié)構(gòu)分析、氨基酸序列結(jié)構(gòu)域和功能預(yù)測及進化分析等生物信息學(xué)分析,結(jié)果表明:1)ChaTHL1和ChaTHL2所編碼的蛋白均為穩(wěn)定性蛋白、其存在一個thioredoxin結(jié)構(gòu)域、具有蛋白質(zhì)二硫氧化還原酶活性,在進化中屬于THL類蛋白;2)ChaMLPK為堿性親水性穩(wěn)定蛋白、富含磷酸化位點(53個)、具有一個STKs結(jié)構(gòu)域,與核桃(Juglans regia)的蛋白激酶APK 1A序列親緣關(guān)系最近;3)ChaARC1為酸性親水性穩(wěn)定蛋白,具有泛素化轉(zhuǎn)移酶活性、存在一個U-box和兩個ARM結(jié)構(gòu)域,與核桃含有U-box結(jié)構(gòu)域的蛋白序列親緣關(guān)系最近;4)ChaExo70A1為堿性親水蛋白、存在一個Exo結(jié)構(gòu)域、屬于胞外分泌復(fù)合體,與核桃Exo70A1親緣關(guān)系最近;5)Cha MOD1和ChaMOD2均為堿性疏水蛋白,屬于PIP家族,是膜的組成部分、參與物質(zhì)轉(zhuǎn)運過程;6)ChaKAPP為酸性親水蛋白、具有磷蛋白磷酸酶活性、存在一個FHA和一個PP2C結(jié)構(gòu)域,與核桃PP2C 70序列親緣關(guān)系最近。以本研究篩選所得的穩(wěn)定基因(ChaActin和ChaEF1-a)作為雙內(nèi)參基因,利用熒光定量PCR對上述基因進行了表達分析,結(jié)果表明:1)ChaMLPK和ChaMOD1/ChaMOD2在花粉中幾乎無表達,但在其他組織中均有表達,其余5個基因為組成型表達,在各組織器官中均有一定量的表達存在;2)在自交不親和授粉后24小時內(nèi)ChaTHL1/ChaTHL2、ChaARC1和ChaKAPP均在2 h時表達量達到最高,而ChaMLPK,ChaMOD1和ChaExo70A1在24 h時達到最高,ChaMOD2則在1 h時達到最高值。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S664.4

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