光周期响应基因StH2A调控生长功能研究

发布时间：2018-07-25 20:54

【摘要】：不同植物对光周期响应不同,目前关于植物如何在分子水平响应光周期知之甚少。本论文以不同光周期处理的马铃薯叶片为材料,转录组测序获得了不同光周期处理的转录组表达谱;分析了表达谱数据,识别和筛选了光周期响应基因,克隆了光周期响应基因StH2A的全长cDNA,基因工程途径实现了反义StH2A cDNA在本氏烟体内表达,确定了 H2A参与顶端优势维持和调控开花的功能,具体研究结果如下:1.经转录组测序、GO功能注释和差异表达基因分析,筛选了 208个注释为“核酸结合蛋白”的差异基因。结合不同光周期这些基因的RPKM表达量变化,筛选了 20个显著随光周期变化表达的目的基因,qRT-PCR证实了其随光周期的表达动态,由此确定了对注释为H3.2、H2A和TP的基因进行进一步研究。2.克隆了H3.2、H2A、TP基因的全长cDNA;为了实现各自编码基因体内过量或抑制表达,亚克隆途径,得到基因工程重组菌株 DH5α-pC-35S-H3.2+/-、DH5α-pC-35S-H2A+/-、DH5α-pC-35S-TP+/-及GV3101-pC-35S-H3.2+/-、GV3101-pC-35S-H2A+/-、GV3101-pC-35S-TP+/-。3.采用根癌农杆菌介导途径,转化反义StH2A于本氏烟,筛选获得本氏烟突变株系h2a-2,h2a-2表现出顶端优势缺失和开花延迟的表型;H2A蛋白ELISA测定结果显示,h2a-2株系叶片H2A蛋白含量比WT下降23.7%,表明反义StH2A本氏烟体内表达抑制了本氏烟NbH2A的积累。4.本氏烟基因组数据库检索,识别了CONSTANS(CO),FLOWING LOCUS T(FT)和EARLY FLOWERING 4(LF4)3个成花基因,依据其核酸序列设计各自特异引物,叶片mRNA为模版,RT-PCR结果显示,与对照株系比较,h2a-2株系3个成花基因mRNA积累呈现不同程度的下调表达,表明NbH2A参与了 3个成花基因的转录,CO,FT与ELF4间通过H2A彼此联系。5.检索本氏烟基因组数据库,识别了 22个NbH2A编码基因,序列对比和进化树分析发现,其中4个NbH2A与本研究克隆的StH2A预测的氨基酸序列高度相似,且具有N端多聚丙氨酸基序,反义StH2A至少抑制了 N端多聚丙氨酸NbH2A的表达。6.不同NbH2A基因RT-PCR结果显示,h2a-2株系叶片多聚丙氨酸NbH2A mRNA积累明显少于对照,其它非多聚丙氨酸NbH2A基因mRNA积累明显多于对照,表明其它NbH2A基因在转录水平互补了多聚丙氨酸NbH2A的表达下调。结论认为:反义StH2A在本氏烟体内表达,抑制了其多聚丙氨酸NbH2A基因mRNA积累,尽管其它非多聚丙氨酸NbH2A mRNA积累增加,并未能弥补烟草H2A蛋白积累的减少;烟草H2A蛋白积累减少,导致顶端优势缺失和开花推迟的表型,说明编码多聚丙氨酸的NbH2A基因正向调控本氏烟的开花和生长。
[Abstract]:The response of different plants to photoperiod is different, and little is known about how plants respond to photoperiod at molecular level. In this paper, the transcriptome expression profiles of potato leaves treated with different photoperiod were obtained by transcriptome sequencing, and the data of expression profiles were analyzed, and the photocycle-responsive genes were identified and screened. The full-length cDNA of photoperiodic response gene (StH2A) was cloned. The antisense StH2A cDNA was expressed in the tobacco by genetic engineering pathway, and the function of H2A in maintaining and controlling flowering was confirmed. The results are as follows: 1. By sequencing go functional annotation and differential expression gene analysis, 208 differentially expressed genes annotated as "nucleic acid binding protein" were screened. In combination with the changes of RPKM expression of these genes in different photoperiod, 20 target genes were screened and their expression dynamics with photoperiod were confirmed by qRT-PCR. Therefore, further studies on the genes annotated as H3.2, H2A and TP have been carried out. The full-length cDNAof H3.2H2Atp gene was cloned, and the recombinant strain DH5 伪 -pC-35S-H3.2 / -DH5 伪 -pC-35S-H2A 伪 -pC-35S-TP /-and GV3101-pC-35S-H3.2 / -GV3101-pC-35S-H2A / -GV3101-pC-35S-TP / -3.3in order to achieve excessive or inhibitory expression in vivo were obtained from the recombinant strain DH5 伪 -pC-35S-H3.2% -pC-35S-H3.2 伪 -pC-35S-H2A 伪 -pC-35S-H2A-pC-35S-TP / -3. Agrobacterium tumefaciens mediated transformation of antisense StH2A into Bentner's tobacco was carried out by Agrobacterium tumefaciens. The phenotypic H2A protein ELISA analysis showed that the H2A protein content in leaves of H2a-2a-2 mutant was lower than that of WT, indicating that the in vivo expression of antisense StH2A inhibited the accumulation of NbH2A. 4. Three floral genes of CONSTANS (CO) flushing LOCUS T (FT) and EARLY FLOWERING 4 (LF4) were identified and their specific primers were designed according to their nucleic acid sequences. The results of RT-PCR showed that mRNA in leaves was a template. Compared with the control lines, the mRNA accumulation of three floral genes of H2a-2 strain was down-regulated in varying degrees, indicating that NbH2A was involved in the transcription of three floral genes, Cocoft and ELF4 were related to each other through H2A. 5. 22 NbH2A coding genes were identified by searching the database of tobacco genome. Sequence comparison and phylogenetic tree analysis showed that 4 of them were highly similar to the predicted amino acid sequences of the cloned StH2A and had N-terminal polyalanine motifs. Antisense StH2A inhibited at least the expression of N terminal polyalanine NbH2A. The results of RT-PCR of different NbH2A genes showed that the accumulation of polyalanine NbH2A mRNA in leaves of H2a-2 strains was significantly lower than that of the control, and the mRNA accumulation of other non-polyalanine NbH2A genes was significantly higher than that of the control. These results suggest that other NbH2A genes complement each other in down-regulation of polyalanine NbH2A expression at the transcriptional level. Conclusion: the expression of antisense StH2A in tobacco inhibits the mRNA accumulation of polyalanine NbH2A gene, although the accumulation of other non-polyalanine NbH2A mRNA increases, but it can not make up for the decrease of H2A protein accumulation and H2A protein accumulation in tobacco. The phenotypes of apical dominance loss and flowering delay indicate that the NbH2A gene encoding polyalanine positively regulates the flowering and growth of Bentworth tobacco.
【学位授予单位】：宁夏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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