本文選題：沙田柚 + 轉(zhuǎn)錄組測序��； 參考：《廣西師范大學(xué)》2017年碩士論文

【摘要】：沙田柚(Citrus grandis var.shatinyu)是廣西的名優(yōu)水果之一,因其品質(zhì)優(yōu)良和較高的營養(yǎng)價(jià)值而廣受人們的歡迎。但是,沙田柚屬于高度配子體自交不親和的果樹,自然坐果率很低,因而要依靠人工異花授粉來提高產(chǎn)量。本研究以未授粉、自交和異交1d、2d、3d的花柱為試材,提取RNA,構(gòu)建cDNA文庫,運(yùn)用第二代高通量測序技術(shù)對其進(jìn)行轉(zhuǎn)錄組測序。隨后,對測序結(jié)果進(jìn)行GO功能注釋、Pathway通路分析和表達(dá)量計(jì)算,從中選擇出可能與沙田柚自交不親和有關(guān)的核糖核酸酶基因 Unigene34907_All(cgSLA)和 Unigene2441_All(cgSRA),構(gòu)建植物表達(dá)載體,轉(zhuǎn)入擬南芥,對其基因功能進(jìn)行驗(yàn)證。本論文的結(jié)果如下:1.樣品通過提取RNA,構(gòu)建cDNA文庫,最終得到總Unigene 90214個(gè),總長142142835nt,平均長度 1576nt,N50 長度為 2413nt。2.獲得的Unigene與NR、NT、Swiss-Prot、KEGG、COG、GO數(shù)據(jù)庫比對后,分別有62679、59673、41897、39362、28317、50701條比對到該數(shù)據(jù)庫。通過COG功能分類,獲得的Unigene被歸類到25個(gè)功能類別。其中被歸類到通用功能、轉(zhuǎn)錄相關(guān)功能的Unigene數(shù)目最多。3.GO功能富集分析和Pathway代謝通路富集分析的結(jié)果表明,獲得的Unigene分別被歸類到55個(gè)GO功能類別和128個(gè)代謝通路。CDs預(yù)測共獲得了62752條CDs,其中比對六大數(shù)據(jù)庫獲得61707條,而用軟件ESTScan預(yù)測得1045條。4.通過SSR分析共獲得29055個(gè)SSR,其中單堿基重復(fù)最多,共有12746個(gè)。通過SNP分析,在7個(gè)樣品中,未授粉花柱的SNP數(shù)為47145;異交1d花柱為53617,異交2d花柱為54143,異交花柱為52717;自交1-3d花柱分別為50606、54005和53816個(gè)。5.從自交和異交花柱中篩選到差異表達(dá)的核糖核酸酶基因38個(gè)。從中選擇了可能與沙田柚自交不親和有關(guān)的兩個(gè)基因Unigene34907_All(cgSLA),Unigene2441_All(cgSRA)。構(gòu)建植物表達(dá)載體,利用花絮浸染法轉(zhuǎn)化擬南芥,PCR和測序的結(jié)果表明基因已整合到擬南芥基因組。6.RT-PCR驗(yàn)證結(jié)果表明,8個(gè)沙田柚花柱差異表達(dá)的表達(dá)趨勢與轉(zhuǎn)租測序結(jié)果相同。7.對自交和異交花柱轉(zhuǎn)錄組測序所得基因進(jìn)行差異表達(dá)分析發(fā)現(xiàn),這些基因主要與植物激素的信號(hào)傳導(dǎo)途徑,或脅迫應(yīng)答反應(yīng)有關(guān)。
[Abstract]:Citrus grandis var. shatinyuis is one of the most famous fruits in Guangxi, which is popular because of its excellent quality and high nutritional value. However, Shatian pomelo is a high gametophyte self-incompatibility fruit tree, the natural fruit setting rate is very low, so it depends on artificial cross-pollination to improve the yield. In this study, the unpollinated, self-bred and heterozygous styles were used as the test materials, RNAs were extracted, cDNA library was constructed, and the second generation high-throughput sequencing technique was used to sequence the transcriptome. Then, the sequencing results were analyzed by go functional annotation Pathway pathway and the expression amount was calculated, from which the ribonuclease genes Unigene34907, AllhcgSLAand Unigene2441AllCgSRAA were selected, and the plant expression vector was constructed and transferred into Arabidopsis thaliana, which may be related to the self-incompatibility of Shatian pomelo, and the ribonuclease gene Unigene34907, which may be related to self-incompatibility, was constructed and transformed into Arabidopsis thaliana. Its gene function was verified. The results of this thesis are as follows: 1: 1. The cDNA library was constructed by RNA extraction, and the total Unigene 90214, the total length was 142142835nt.The average length of N50 was 2413nt.2. After the comparison of Unigene with NRN NTS Swiss-ProtKEGGGG go database, there were 62679 / 59673 / 41897 / 39362 / 283171750701 and 62679 / 59673 / 596 / 701, respectively. The obtained Unigene is classified into 25 functional categories by COG functional classification. The results of functional enrichment analysis of Unigene and Pathway metabolic pathway showed that the number of Unigene with transcription related function was the highest. 3. Unigene was classified into 55 go functional categories and 128 metabolic pathways. CDs predicted 62752 CDss, of which 61707 were compared with six databases and 1045 by software ESTScan. A total of 29055 SSRs were obtained by SSR analysis, of which 12746 were single base repeats. According to SNP analysis, the number of SNP of unpollinated style was 47145. the number of SNP was 53617 in 1 d, 54143 in 2d, 52717 in heterozygosity, 560654005 in self-pollination and 53816. 5 in self-pollination. 38 differentially expressed ribonuclease genes were screened from self-and heterozyme styles. Two genes, Unigene 34907, which may be related to the self-incompatibility of Shaddock, were selected. Plant expression vector was constructed and transformed into Arabidopsis thaliana by PCR and sequencing. The results showed that the gene had been integrated into Arabidopsis thaliana genome. 6. RT-PCR results showed that the differential expression trend of 8 Shatian pomelo styles was the same as that of sublet sequencing. The differentially expressed genes obtained from transcriptome sequencing of self-and heterozygous styles showed that these genes were mainly related to the signal transduction pathway of plant hormones or the response to stress.
【學(xué)位授予單位】：廣西師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S666.3

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