羊绒周期性生长特异性IncRNAs的筛选

发布时间：2018-12-17 07:38

【摘要】：长链非编码RNAs(long noncoding RNA,lnc RNAs)是一类本身不编码蛋白、转录本长度超过200 nt的RNA分子,它可以在表观遗传调控、转录调控以及转录后调控等多种层面上调控基因的表达。由于许多lnc RNAs仅在特定的发育阶段出现,而且羊绒的生长具有周期性的特点,因此本研究利用RNA-seq技术,对羊绒生长期和休止期的皮肤组织分别进行de novo测序及生物信息学分析,通过对羊绒生长期和休止期显著差异表达lnc RNAs的筛选及q RT-PCR验证,对其中的5个lnc RNAs构建了过表达载体,为进一步研究lnc RNAs的功能和探讨特异性lnc RNAs在羊绒生长中的作用提供理论依据。获得的结果如下:1.RNA-seq的结果分析:RNA-seq得到的转录组的reads数量在生长期和休止期分别为51531778和61750544,表达的转录本数量为37574和37619;两个时期表达的基因数量为37649,其中差异表达的基因数量为977个(P0.05),表达上调的基因有559个,下调的基因有418个;两个时期表达的lnc RNAs的数量是6127个,只有54个lnc RNAs差异表达,其中上调的32个,下调的为22个。GO分析结果表明,这些转录本在分子功能,细胞组成和生物学进程中具有重要作用,包括Hox A、Hox B、Hox C和Hox D家族基因,BMPs家族以及KAPs、FGFs基因及其受体基因等。KEGG的分析结果表明有些lnc RNAs与信号传导机制相关。2.利用q RT-PCR对10个生长期和休止期显著差异表达的lnc RNAs进行验证,结果表明:这10个lnc RNAs相对表达量的q RT-PCR结果与RNA-seq测序结果的趋势一致,但是lnc RNA9686的差异不显著。3.对于验证的lnc RNA9686、lnc RNA14672、lnc RNA19457、lnc RNA15479和lnc RNA13025,用p B-CMV-Ds Red-CMV-GFP.BS.with repeats的骨架载体利用PCR及双酶切连接的方法构建过表达载体,通过双酶切及测序验证,表明过表达载体构建成功,为进一步研究lnc RNAs的功能奠定了基础。
[Abstract]:Long chain noncoding RNAs (long noncoding RNA,lnc RNAs) is a class of RNA molecules which do not encode proteins and whose transcripts are more than 200 nt in length. They can regulate the expression of genes at many levels, such as epigenetic regulation, transcriptional regulation and posttranscriptional regulation. Due to the fact that many lnc RNAs only occur at specific developmental stages and the growth of cashmere is periodic, the RNA-seq technique is used in this study. De novo sequencing and bioinformatics analysis were performed on the skin tissues of cashmere during growth period and rest period respectively. The expression of lnc RNAs was screened and verified by Q RT-PCR. The overexpression vectors of five of them were constructed to provide theoretical basis for further study of the function of lnc RNAs and the role of specific lnc RNAs in cashmere growth. The results were as follows: the results of 1.RNA-seq analysis showed that the reads number of the transcriptome obtained by RNA-seq was 51531778 and 61750544 during the growth period and the rest period, and the number of expressed transcripts was 37574 and 37619 respectively. The number of genes expressed in the two periods was 37649, among which 977 genes were differentially expressed (P0.05), 559 genes were up-regulated and 418 genes were down-regulated. The number of lnc RNAs expressed in the two periods was 6127, with only 54 lnc RNAs differentially expressed, of which 32 were up-regulated and 22 were down-regulated. GO analysis showed that these transcripts were functionally active. Cell composition and biological processes play an important role, including Hox, Hox C and Hox D genes, BMPs family, and KAPs, FGFs gene and its receptor gene, etc. The results of KEGG analysis showed that some lnc RNAs were related to signal transduction mechanism. 2. Q RT-PCR was used to verify the expression of 10 lnc RNAs with significant difference in growth period and rest period. The results showed that the Q RT-PCR results of the relative expression of 10 lnc RNAs were consistent with the results of RNA-seq sequencing. But the difference in lnc RNA9686 was not significant. For the verified lnc RNA9686,lnc RNA14672,lnc RNA19457,lnc RNA15479 and lnc RNA13025, the overexpression vector was constructed by using the framework vector of p B-CMV-Ds Red-CMV-GFP.BS.with repeats by PCR and double enzyme ligation, and the expression vector was confirmed by double enzyme digestion and sequencing. The results showed that the overexpression vector was successfully constructed, which laid a foundation for further study on the function of lnc RNAs.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827

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