沙田柚自交和异交花柱转录组测序及核糖核酸酶基因（cgSLA、cgSRA）的克隆与表达分析

发布时间：2018-06-16 00:25

本文选题：沙田柚 + 转录组测序　；参考：《广西师范大学》2017年硕士论文

【摘要】：沙田柚(Citrus grandis var.shatinyu)是广西的名优水果之一,因其品质优良和较高的营养价值而广受人们的欢迎。但是,沙田柚属于高度配子体自交不亲和的果树,自然坐果率很低,因而要依靠人工异花授粉来提高产量。本研究以未授粉、自交和异交1d、2d、3d的花柱为试材,提取RNA,构建cDNA文库,运用第二代高通量测序技术对其进行转录组测序。随后,对测序结果进行GO功能注释、Pathway通路分析和表达量计算,从中选择出可能与沙田柚自交不亲和有关的核糖核酸酶基因 Unigene34907_All(cgSLA)和 Unigene2441_All(cgSRA),构建植物表达载体,转入拟南芥,对其基因功能进行验证。本论文的结果如下:1.样品通过提取RNA,构建cDNA文库,最终得到总Unigene 90214个,总长142142835nt,平均长度 1576nt,N50 长度为 2413nt。2.获得的Unigene与NR、NT、Swiss-Prot、KEGG、COG、GO数据库比对后,分别有62679、59673、41897、39362、28317、50701条比对到该数据库。通过COG功能分类,获得的Unigene被归类到25个功能类别。其中被归类到通用功能、转录相关功能的Unigene数目最多。3.GO功能富集分析和Pathway代谢通路富集分析的结果表明,获得的Unigene分别被归类到55个GO功能类别和128个代谢通路。CDs预测共获得了62752条CDs,其中比对六大数据库获得61707条,而用软件ESTScan预测得1045条。4.通过SSR分析共获得29055个SSR,其中单碱基重复最多,共有12746个。通过SNP分析,在7个样品中,未授粉花柱的SNP数为47145;异交1d花柱为53617,异交2d花柱为54143,异交花柱为52717;自交1-3d花柱分别为50606、54005和53816个。5.从自交和异交花柱中筛选到差异表达的核糖核酸酶基因38个。从中选择了可能与沙田柚自交不亲和有关的两个基因Unigene34907_All(cgSLA),Unigene2441_All(cgSRA)。构建植物表达载体,利用花絮浸染法转化拟南芥,PCR和测序的结果表明基因已整合到拟南芥基因组。6.RT-PCR验证结果表明,8个沙田柚花柱差异表达的表达趋势与转租测序结果相同。7.对自交和异交花柱转录组测序所得基因进行差异表达分析发现,这些基因主要与植物激素的信号传导途径,或胁迫应答反应有关。
[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.
【学位授予单位】：广西师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S666.3

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