雷竹SVP-like、SOC1-like基因的克隆及功能分析
發(fā)布時(shí)間：2018-11-24 07:49
【摘要】：竹子開(kāi)花的周期長(zhǎng)、時(shí)間不確定,一般3-120年,而且大部分竹種開(kāi)花后死亡,這種開(kāi)花的獨(dú)特性限制了竹子開(kāi)花的研究。此外,開(kāi)花后的成片死亡導(dǎo)致整個(gè)竹林衰敗,并造成嚴(yán)重的經(jīng)濟(jì)損失。因此,研究竹子開(kāi)花具有重要的理論和經(jīng)濟(jì)價(jià)值。通過(guò)對(duì)擬南芥(Arabidopsis thaliana)的研究發(fā)現(xiàn),SHORT VEGETATIVE PHASE (SVP)和SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1)是重要開(kāi)花整合因子。本論文利用同源克隆技術(shù)從雷竹(Phyllostachys violasces)中克隆SVP和SOC1的同源基因并進(jìn)行功能分析。主要的研究結(jié)果如下：1.從雷竹中分離出兩個(gè)SVP-like和一個(gè)SCC1-like基因,分別命名為PvSVP1、PvSVP2和PvMADS56。2.序列比對(duì)和進(jìn)化樹(shù)分析發(fā)現(xiàn),PvSVP1、PvSVP2與禾本科中的SVP-like蛋白聚為一類,其中PvSVP1與OsMADS55的關(guān)系最近,而PvSVP2與OsMADS47的關(guān)系最近。組織特異性表達(dá)分析表明,PvSVP1和PvSVP2在開(kāi)花和不開(kāi)花雷竹的各個(gè)組織中都有表達(dá)；開(kāi)花雷竹中,它們?cè)诙捄椭癖拗械谋磉_(dá)量較高；不開(kāi)花雷竹中,PvSVP1在嫩葉中的表達(dá)量最高,而PvSVP2在竹鞭中的表達(dá)量最高。隨著花的發(fā)育二者表現(xiàn)出不同的表達(dá)趨勢(shì),PvSVP1的表達(dá)水平表現(xiàn)為先下降后上升,而PvSVP2的表達(dá)水平則是先上升后保持穩(wěn)定。轉(zhuǎn)化擬南芥實(shí)驗(yàn)發(fā)現(xiàn),在長(zhǎng)日照和短日照條件下,35S::PvSVPl轉(zhuǎn)基因植株均提早開(kāi)花并產(chǎn)生不正常的花器官。RT-qPCR檢測(cè)轉(zhuǎn)基因擬南芥中開(kāi)花相關(guān)基因的表達(dá),結(jié)果表明在長(zhǎng)日照條件下PvSVPl通過(guò)間接調(diào)控FT、FLC、AP1、PI的表達(dá)影響擬南芥開(kāi)花時(shí)間及參與花器官發(fā)育。轉(zhuǎn)化水稻(Oryza sativa)實(shí)驗(yàn)發(fā)現(xiàn),與野生型比較PvSVP1的過(guò)表達(dá)使水稻開(kāi)花時(shí)間發(fā)生改變；轉(zhuǎn)基因植株在短日照條件下表現(xiàn)為早花,但在長(zhǎng)日照條件下卻表現(xiàn)為晚花。RT-qPCR結(jié)果表明在長(zhǎng)日照條件下PvSVP1的過(guò)表達(dá)間接地下調(diào)Hd3a、RFT1、OsMADS50的表達(dá)水平,同時(shí)上調(diào)了Ghd7表達(dá)水平,從而抑制水稻的開(kāi)花；短日照條件下,PvSVP1的過(guò)表達(dá)間接下調(diào)Ghd7的表達(dá)量,同時(shí)上調(diào)Hd3a、RFT1的表達(dá)量,進(jìn)而使水稻提早開(kāi)花。此外35S::PvSVP1轉(zhuǎn)擬南芥和水稻植株的株高產(chǎn)生變化。35S:. PvSVP2轉(zhuǎn)基因擬南芥也產(chǎn)生早花和不正�；ㄆ鞴俚谋硇汀vSVP1、PvSVP2主要定位于細(xì)胞核中,酵母雙雜交及雙熒光互補(bǔ)實(shí)驗(yàn)證明它們與PvAP1、PvVRN1、PvMADS56、PvSEP3存在相互作用。原核表達(dá)分析驗(yàn)證這兩個(gè)蛋白是可溶的。以上結(jié)果說(shuō)明PvSVP1、PvSVP2在參與花發(fā)育方面與其它SvP-like基因功能相似,但在調(diào)控開(kāi)花時(shí)間方面可能存在差異。3.序列比對(duì)和進(jìn)化樹(shù)分析發(fā)現(xiàn)PvMADS56屬于SOC1-like家族中MADS56-like亞分支。PvMADS56在開(kāi)花和不開(kāi)花雷竹的各個(gè)部位中都有表達(dá)。轉(zhuǎn)基因?qū)嶒?yàn)表明PvMADS56的過(guò)表達(dá)能使擬南芥早花并能互補(bǔ)socl突變體的晚花表型；此外轉(zhuǎn)基因植株產(chǎn)生了不正常的花器官和葉片、低結(jié)實(shí)率和株高變矮的表型。RT-qPCR實(shí)驗(yàn)結(jié)果表明PvMADS56的過(guò)表達(dá)通過(guò)上調(diào)FT和下調(diào)FLC促使轉(zhuǎn)野生型擬南芥植株早花,同時(shí)調(diào)控AP1、AP3、PI的表達(dá)水平參與花器官的發(fā)育。PvMADS56是一個(gè)核蛋白,與PvAP1、PvSEP3存在相互作用。此外PvMADS56的啟動(dòng)子活性受ABA和MeJA影響。以上結(jié)果說(shuō)明PvMADS56可能是多功能基因,它可能響應(yīng)ABA和MeJA調(diào)控開(kāi)花時(shí)間和花器官的發(fā)育。本文的研究為揭示竹子開(kāi)花的分子機(jī)理提供了理論依據(jù)。
[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.
【學(xué)位授予單位】：北京林業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S795
本文編號(hào)：2352858
[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.
【學(xué)位授予單位】：北京林業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S795
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