本文關鍵詞： 蓮 美洲黃蓮 花色 花青素 MYB GST　出處：《中國科學院研究生院(武漢植物園)》2016年博士論文　論文類型：學位論文

【摘要】：蓮是一種集觀賞、藥用和食用于一體的水生植物。蓮屬包含兩個物種,即蓮(Nelumbo nucifera Gaertn.)和美洲黃蓮(N. lutea Pers.)。兩物種雖然間斷分布于東亞-北美和澳大利亞北部,但是不存在生殖隔離,雜交可育。除此之外,兩物種在形態(tài)上差異不明顯,最顯著的差異是花色。蓮具有紅色、粉色、白色和紅白相間四種花色,獨缺黃色,而美洲黃蓮只有黃色。有關蓮屬花色的分子機理非常少,目前僅有紅、白花色差異的研究,沒有兩物種間花色差異的研究。究竟是什么導致了親緣關系相近的蓮與美洲黃蓮在花色上差異如此顯著?為了解開這個謎底,本研究以黑龍江的野生蓮(紅色花)和美國佛羅里達州的野生美洲黃蓮(黃色花)為材料進行研究。首先,本研究采用分光光度計法測量了紅、黃兩種花瓣中花青素的含量。然后,通過基因克隆、本地blast和系統(tǒng)發(fā)育樹的構(gòu)建,找到在蓮屬中參與花青素合成及轉(zhuǎn)運的結(jié)構(gòu)基因和調(diào)節(jié)基因。通過比較這些基因在兩物種中的核苷酸序列及轉(zhuǎn)錄水平,找到可能參與花色差異的候選基因,并結(jié)合酵母雙雜和轉(zhuǎn)基因技術進行驗證。取得的主要結(jié)果如下：(1)野生蓮的紅色花瓣中花青素的含量較高,而美洲黃蓮的黃色花瓣中幾乎不含花青素。這表明美洲黃蓮花瓣中花青素合成途徑可能發(fā)生中斷。波長掃描顯示在350nm處,兩種花色的花瓣提取物都有一個高的吸收峰,表明黃酮和黃酮醇存在于這兩種提取物中。鑒于黃酮和黃酮醇是在花青素合成途徑的中間步驟合成的,因此我們推測美洲黃蓮中花青素合成途徑的上游基因是有功能的,中斷可能發(fā)生在花青素合成途徑的下游。(2)通過簡并PCR技術,從兩物種中獲得下游基因DFR1和ANS1�；谏徎蚪M的信息,我們構(gòu)建本地blast。通過進行本地blast:和構(gòu)建系統(tǒng)發(fā)育樹,在蓮中鑒定到參與花青素合成的4個UFGTs(UGT78A1-A4)和2個GSTs(GSTF11和GSTF12)基因。以紅色花瓣的cDNA為模板進行擴增,檢測到了4個UFGT的轉(zhuǎn)錄本和1個GST基因(GSTF11)的轉(zhuǎn)錄本。通過在兩物種中比較這些結(jié)構(gòu)基因的核苷酸序列及推測的氨基酸序列,發(fā)現(xiàn)這些結(jié)構(gòu)基因具有較高的同源性,而且在已知的保守區(qū)和保守位點中幾乎沒有氨基酸差異。通過熒光實時定量PCR,發(fā)現(xiàn)DFR1、ANSI和UGT78A2在美洲黃蓮的黃色花瓣中的表達水平高于蓮紅色花瓣中的表達水平,而剩下的3個UFGT基因在美洲黃蓮花瓣中的表達水平低于蓮花瓣中的表達水平。半定量PCR顯示蓮的花瓣中存在GSTs的轉(zhuǎn)錄本,而美洲黃蓮的花瓣中不存在GSTs基因的轉(zhuǎn)錄本。GSTs基因轉(zhuǎn)錄本的缺失可能是美洲黃蓮花瓣中不含花青素的關鍵原因。(3)通過本地blast,從蓮基因組中總共鑒定到95個MYB基因,10個bHLH基因和17個WD40基因。通過與其他物種中已報道參與花青素合成的調(diào)節(jié)基因進行對比并構(gòu)建系統(tǒng)發(fā)育樹,我們發(fā)現(xiàn)蓮中有9個MYB基因,2個bHLH基因和1個WD40基因(即TTG1)基因可能參與花青素合成途徑的調(diào)控。以紅色花瓣的cDNA為模板,通過PCR擴增,我們鑒定到1個MYB基因(即MYB5)和1個bHLH基因(即bHLH1)參與花瓣中花青素合成的調(diào)控。序列比較顯示,在兩物種中TTG1的氨基酸序列是一致的,而bHLH1只有1個氨基酸差異發(fā)生在bHLH結(jié)構(gòu)域中。最顯著的差異是MYB5基因,由于兩個氨基酸的替代形成了一個提前終止密碼子。對啟動子區(qū)域潛在的順式作用元件進行預測,結(jié)果顯示美洲黃蓮MYB5啟動子有14種順式作用元件,而蓮MYB5中有13種。兩物種的MYB5基因啟動子中都包含很多涉及光響應的順式作用元件。(4)居群水平上的研究揭示了MYB5基因有3種類型的等位基因。一種為有功能的等位基因,以純合子的形式存在于蓮的8個居群中；第二種為兩個核苷酸替代(GAA/TAG)導致形成了一個提前終止密碼子；第三種為一個核苷酸缺失導致形成了一個提前終止密碼子。這兩種無功能的等位基因只存在于美洲黃蓮的居群中。其中第二種無義突變的等位基因存在于美洲黃蓮的8個居群中,而第三種移碼突變的等位基因存在于美洲黃蓮的6個居群中。無義突變的等位基因有純合子形式,而移碼突變的等位基因總是和無義突變的等位基因一起,以雜合子形式存在,尚未檢測到純合子形式。(5)酵母雙雜實驗證明,蓮中的NnMYB5不僅可以與蓮中的NnbHLH1及NnTTG1互作,也可以與美洲黃蓮中的N1bHLH1相互作用。而蓮中NnTTGl也可以與兩物種中的bHLH1相互作用。以上結(jié)果暗示,MYB5、bHLH1和TTG1可以形成MBW三元復合體。NnMYB5與NnbHLH1的相互作用強于與N1bHLH1的相互作用,表明bHLH1在兩物種中的差異影響了其與MYB5的互作。(6)NnMYB5基因在擬南芥中的過表達導致花青素在未成熟的種子及花梗中積累,表明NnMYB5是花青素合成的轉(zhuǎn)錄激活子。擬南芥AtGsTF12基因在NnMYB5過表達植株中表達水平上調(diào),表明AtGsTF12基因是NnMYB5的靶基因。由上述結(jié)果,推測出以下結(jié)論：蓮與美洲黃蓮的紅、黃花色差異可能是由于兩物種中的MYB5基因編碼區(qū)的突變影響了其對結(jié)構(gòu)基因的調(diào)節(jié),進而影響了結(jié)構(gòu)基因的表達水平與花青素的穩(wěn)定積累所致。
[Abstract]:Lotus is a kind of ornamental, medicinal and edible aquatic plants. In one of the genus contains two species, namely the lotus (Nelumbo nucifera Gaertn.) and American Huang Lian (N. lutea Pers.). Although the two species discontinuously distributed in East Asia and North America and northern Australia, but there is no reproductive isolation, the hybrids. Besides, two species in the form of difference is not obvious, the most significant difference is the color. Lotus with red, pink, white and red and white four colors, no yellow, yellow. And America Huang Lian only molecular mechanism about the color of the lotus is very small, the only red, white color difference study, no differences the two flower species. What is the lotus and America Huang Lian what caused the close relationship in the color difference is so significant? In order to solve the mystery. In this study, the Heilongjiang wild lotus (red flower) and the American state of Florida Wild goldenseal (yellow flower) were studied. Firstly, spectrophotometer measurement red this study adopts the anthocyanin content in petals of yellow two. Then, through gene cloning, phylogenetic tree construction and local blast system, find the structural genes involved in anthocyanin synthesis and transport in the regulation and. The gene. By comparing nucleotide sequences of these genes in two species and the level of transcription, finding candidate genes may be involved in color difference, which is verified by yeast two hybrid and transgenic technology. The main results are as follows: (1) high content of anthocyanins in the wild lotus petals red, and yellow petals in goldenseal almost does not contain anthocyanin. This indicates that anthocyanin synthesis pathway in goldenseal petals could break. The wavelength scanning is displayed in the 350nm, two kinds of color of the petal extract has a high absorption Collect peak showed that flavones and flavonols exist in these two kinds of extracts. In view of flavonoids and flavonols are synthesized in the intermediate step of the anthocyanin synthesis pathway, so we speculate that the upstream genes of anthocyanin biosynthesis in goldenseal is a function of the interruption may occur in the anthocyanin synthesis pathway (2). By degenerate PCR, obtain the downstream gene DFR1 and ANS1. from two species of lotus genome based on information, we construct the local blast. through the local blast: and the phylogenetic tree, in the lotus 4 to participate in the identification of anthocyanin synthesis of UFGTs (UGT78A1-A4) and 2 GSTs (GSTF11 and GSTF12) gene in red. The petals of the cDNA was amplified, detected transcripts of 4 UFGT and 1 GST (GSTF11) gene transcripts. The amino acid sequence of nucleotide sequence comparison in two species of these structural genes and putative, The structure of genes with high homology in the conserved region, and known and conserved sites almost no amino acid difference. By real-time quantitative PCR, DFR1, the expression level of ANSI and UGT78A2 in goldenseal yellow petals in the above expression level in red lotus petals, and the expression level of 3 genes left UFGT in goldenseal petals in the lower expression level of lotus petals. The semi quantitative PCR showed the presence of GSTs transcripts of lotus petals, and goldenseal petals does not exist in the GSTs transcript of.GSTs gene transcript deletion may be a key reason of anthocyanin containing no goldenseal petals (3). Through the local blast, a total of 95 identified MYB gene from Lotus genome, 10 bHLH genes and 17 WD40 genes. With other species have been reported to be involved in the synthesis of anthocyanin regulatory gene for Ratio and phylogenetic tree, we found that there are 9 MYB genes in lotus, 2 bHLH genes and 1 WD40 genes (TTG1) gene may be involved in the biosynthesis of anthocyanins in red petals. CDNA as template, amplified by PCR, we identified 1 MYB genes (MYB5 and 1) a bHLH gene (bHLH1) is involved in the regulation of anthocyanin synthesis in petals. Sequence comparison showed that the amino acid sequence of TTG1 in the two species is the same, but bHLH1 has only 1 amino acid differences occurred in the bHLH domain. The most significant difference is due to the MYB5 gene, two amino acid substitution forms a a premature termination codon. The promoter region of potential cis acting elements were predicted. Results showed that goldenseal MYB5 promoter has 14 cis acting elements, and the lotus MYB5 13 species. Two species of MYB5 gene promoter contains many related CIS light response Effect of element. (4) in the study group on the level of the MYB5 gene revealed 3 types of alleles. A functional allele, YISHION zygote exists in the form of lotus of the 8 populations; second to two nucleotide substitutions (GAA/TAG) leads to the formation of a early termination codon third; a nucleotide deletion leads to the formation of a premature termination codon. These two kinds of function alleles exist only in goldenseal populations. Among the second kinds of nonsense mutation alleles exist in goldenseal 8 populations, and third a frameshift allele is present in goldenseal 6 populations. No allelic mutation is homozygous, and frameshift allele always and nonsense mutant alleles together, exist in the heterozygous form, has not detected the homozygous form (5). Yeast That two hybrid experiments, the lotus NnMYB5 can interact with NnbHLH1 and NnTTG1 in roots, but also with the goldenseal N1bHLH1 interaction. And the lotus NnTTGl can also interact with the two species of bHLH1. These results suggested that MYB5, bHLH1 and TTG1 can form MBW complex with.NnMYB5 three yuan the interaction of NnbHLH1 in strong interaction with N1bHLH1 and bHLH1 in two species showed differences in the effects of interaction with MYB5. (6) NnMYB5 gene over expression in Arabidopsis thaliana resulted in anthocyanin accumulation in immature seeds and pedicel, indicate that NnMYB5 is a transcriptional activator of anthocyanin biosynthesis in Arabidopsis AtGsTF12 gene. Over expression was up-regulated in plants in NnMYB5, suggesting that the AtGsTF12 gene is the target gene of NnMYB5. From the results that the following conclusions: lotus and goldenseal red, yellow color difference may be due to two species The mutation of the MYB5 gene coding region affects the regulation of the structural genes, and then affects the expression level of the structural genes and the stable accumulation of anthocyanins.

【學位授予單位】：中國科學院研究生院(武漢植物園)
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S682.32;Q943.2

【參考文獻】

相關期刊論文 前1條

1 Wang Li;Bing Wang;Man Wang;Min Chen;Jing-Ming Yin;Ghullam Murtaza Kaleri;Rui-Jie Zhang;Tie-Niu Zuo;Xiong You;Qing Yang;;Cloning and characterization of a potato StAN11 gene involved in anthocyanin biosynthesis regulation[J];Journal of Integrative Plant Biology;2014年04期

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本文編號：1493944