雷竹SVP-like、SOC1-like基因的克隆及功能分析

发布时间：2018-11-24 07:49

【摘要】：竹子开花的周期长、时间不确定,一般3-120年,而且大部分竹种开花后死亡,这种开花的独特性限制了竹子开花的研究。此外,开花后的成片死亡导致整个竹林衰败,并造成严重的经济损失。因此,研究竹子开花具有重要的理论和经济价值。通过对拟南芥(Arabidopsis thaliana)的研究发现,SHORT VEGETATIVE PHASE (SVP)和SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1)是重要开花整合因子。本论文利用同源克隆技术从雷竹(Phyllostachys violasces)中克隆SVP和SOC1的同源基因并进行功能分析。主要的研究结果如下：1.从雷竹中分离出两个SVP-like和一个SCC1-like基因,分别命名为PvSVP1、PvSVP2和PvMADS56。2.序列比对和进化树分析发现,PvSVP1、PvSVP2与禾本科中的SVP-like蛋白聚为一类,其中PvSVP1与OsMADS55的关系最近,而PvSVP2与OsMADS47的关系最近。组织特异性表达分析表明,PvSVP1和PvSVP2在开花和不开花雷竹的各个组织中都有表达；开花雷竹中,它们在秆和竹鞭中的表达量较高；不开花雷竹中,PvSVP1在嫩叶中的表达量最高,而PvSVP2在竹鞭中的表达量最高。随着花的发育二者表现出不同的表达趋势,PvSVP1的表达水平表现为先下降后上升,而PvSVP2的表达水平则是先上升后保持稳定。转化拟南芥实验发现,在长日照和短日照条件下,35S::PvSVPl转基因植株均提早开花并产生不正常的花器官。RT-qPCR检测转基因拟南芥中开花相关基因的表达,结果表明在长日照条件下PvSVPl通过间接调控FT、FLC、AP1、PI的表达影响拟南芥开花时间及参与花器官发育。转化水稻(Oryza sativa)实验发现,与野生型比较PvSVP1的过表达使水稻开花时间发生改变；转基因植株在短日照条件下表现为早花,但在长日照条件下却表现为晚花。RT-qPCR结果表明在长日照条件下PvSVP1的过表达间接地下调Hd3a、RFT1、OsMADS50的表达水平,同时上调了Ghd7表达水平,从而抑制水稻的开花；短日照条件下,PvSVP1的过表达间接下调Ghd7的表达量,同时上调Hd3a、RFT1的表达量,进而使水稻提早开花。此外35S::PvSVP1转拟南芥和水稻植株的株高产生变化。35S:. PvSVP2转基因拟南芥也产生早花和不正常花器官的表型。PvSVP1、PvSVP2主要定位于细胞核中,酵母双杂交及双荧光互补实验证明它们与PvAP1、PvVRN1、PvMADS56、PvSEP3存在相互作用。原核表达分析验证这两个蛋白是可溶的。以上结果说明PvSVP1、PvSVP2在参与花发育方面与其它SvP-like基因功能相似,但在调控开花时间方面可能存在差异。3.序列比对和进化树分析发现PvMADS56属于SOC1-like家族中MADS56-like亚分支。PvMADS56在开花和不开花雷竹的各个部位中都有表达。转基因实验表明PvMADS56的过表达能使拟南芥早花并能互补socl突变体的晚花表型；此外转基因植株产生了不正常的花器官和叶片、低结实率和株高变矮的表型。RT-qPCR实验结果表明PvMADS56的过表达通过上调FT和下调FLC促使转野生型拟南芥植株早花,同时调控AP1、AP3、PI的表达水平参与花器官的发育。PvMADS56是一个核蛋白,与PvAP1、PvSEP3存在相互作用。此外PvMADS56的启动子活性受ABA和MeJA影响。以上结果说明PvMADS56可能是多功能基因,它可能响应ABA和MeJA调控开花时间和花器官的发育。本文的研究为揭示竹子开花的分子机理提供了理论依据。
[Abstract]:The long period of the flowering of the bamboo, the time is not determined, usually 3 to 120 years, and most of the bamboo species died after the flowering, and the uniqueness of the blooming limits the research of the flowering of the bamboo. In addition, after flowering, the death of the whole bamboo forest causes the whole bamboo forest to decline and cause serious economic loss. Therefore, it is of great theoretical and economic value to study the flowering of bamboo. By the study of Arabidopsis thaliana, the SHORT VEGETTIVE PHASE (SVP) and the SUPPRESSOR OF OVEREXPRESS OF CO1 (SOC1) are important flowering whole factors. In this paper, the homologous genes of SVP and SOC1 were cloned from Pyllostachys violasces by homologous cloning, and functional analysis was carried out. The main results are as follows: 1. Two SVP-like and one SCC1-like genes were isolated from the bamboo, named PvSVP1, PvSVP2 and PvMADS5.6, respectively. It is found that PvSVP1, PvSVP2 and SVP-like protein in Gramineae are a class, and the relationship between PvSVP1 and OsMADS55 is most recent, and the relationship between PvSVP2 and OsMADS47 is most recent. The analysis of tissue-specific expression indicated that PvSVP1 and PvSVP2 were expressed in each of the tissues of the flowering and non-flowering plants, and the expression of PvSVP1 in the stem and the whip was higher in the flowering and defoliated bamboo, and the expression of PvSVP2 in the tender leaf was the highest, while the expression of PvSVP2 in the bamboo whip was the highest. With the development of flower, the expression level of PvSV1 was first decreased and the expression level of PvSVP2 was stable after the first increase. Transformation of Arabidopsis has found that under long-and short-day conditions, 35S:: PvSVPl transgenic plants are flowering early and produce abnormal flower organs. The expression of flowering-related genes in transgenic Arabidopsis was detected by RT-qPCR, and the results showed that the expression of PvSVPl in the long-day sunlight affected the flowering time of Arabidopsis and the development of flower organs by indirect regulation of FT, FLC, AP1 and PI. The transformation of rice (Oryza sativa) has shown that the over-expression of PvSV1 with the wild-type results in a change in the flowering time of the rice; the transgenic plant appears as an early flower under the short-day sunshine condition, but it appears to be a late flower under long-day sunshine conditions. The results of RT-qPCR indicated that the overexpression of PvSV1 was down-regulated by the overexpression of PvSV1 under the condition of long sunshine, and the expression level of hd3a, RFT1 and OsMADS50 was reduced, and the expression level of Ghd7 was increased, and the expression of hd3a and RFT1 was up-regulated by the overexpression of PvSV1 under the condition of short sunlight. so that the rice can blossom early. In addition, the strain of 35S:: PvSVP1 to Arabidopsis and rice plants has changed. 35S:. The PvSVP2 transgenic Arabidopsis also produces the phenotype of early and non-normal flower organs. PvSVP1 and PvSVP2 are mainly located in the nucleus, and the yeast two-hybrid and double-fluorescence complementary experiments show that they interact with PvAP1, PvVRN1, PvMADS56 and PvSEP3. Prokaryotic expression analysis verifies that both proteins are soluble. The above results show that PvSVP1 and PvSVP2 are similar to other SvP-like gene functions in flower development, but there may be a difference in regulating flowering time. PvMADS56 is found to belong to the MADS56-like subbranch in the SOC1-like family. The PvMADS56 is expressed in the various parts of the flowering and non-flowering plants. The transgenic experiment shows that the overexpression of PvMADS56 enables the early flowering of the Arabidopsis thaliana and the late flower phenotype of the complementary socel mutant; in addition, the transgenic plant has the phenotype of abnormal flower organs and leaves, low seed setting rate and high plant height. The results of RT-qPCR indicated that the overexpression of PvMADS56 resulted in the early flowering of wild-type Arabidopsis plants by up-regulation of FT and down-regulation of FLC, while regulating the expression levels of AP1, AP3, and PI in the development of flower organs. PvMADS56 is a nucleoprotein that interacts with PvAP1, PvSEP3. In addition, the promoter activity of PvMADS56 is affected by ABA and MeJA. The above results indicate that the PvMADS56 may be a multi-functional gene that may respond to ABA and MeJA to regulate the flowering time and the development of the flower organ. The research of this paper provides a theoretical basis for revealing the molecular mechanism of the flowering of bamboo.
【学位授予单位】：北京林业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S795

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