芍藥類黃酮合成相關(guān)基因載體構(gòu)建及對(duì)模式植物轉(zhuǎn)化研究
發(fā)布時(shí)間：2019-01-18 21:09
【摘要】：花卉植物的花色直接影響了其觀賞價(jià)值,是花卉育種的焦點(diǎn)。芍藥(Paeonia lactiflora)屬于芍藥科芍藥屬,是具有可觀的商品價(jià)值的觀賞植物,但其顏色主要集中在粉色、紅色、紫色、白色系,黃色系等品種非常稀少,這在一定程度上限制了其發(fā)展。對(duì)芍藥花色的改良創(chuàng)新一直備受關(guān)注,主要報(bào)道集中在雜交育種,利用基因工程手段進(jìn)行花色定向選育的研究受到限制,可能由于目前對(duì)芍藥花色形成的分子機(jī)理了解甚少,同時(shí)相關(guān)基因的調(diào)節(jié)機(jī)制無深刻的認(rèn)識(shí)。與花色密切相關(guān)的類黃酮合成途徑中的許多關(guān)鍵基因已在許多植物中進(jìn)行了克隆研究,因此,本研究基于課題組前期對(duì)芍藥嵌合體品種花瓣的轉(zhuǎn)錄組測(cè)序結(jié)果,在明確CHI、ANS、DFR、FLS、PAL基因均為調(diào)控芍藥類黃酮合成途徑中調(diào)控花色的關(guān)鍵基因之后,克隆獲得目的基因,并構(gòu)建這五個(gè)基因的表達(dá)載體,以農(nóng)桿菌為介導(dǎo)轉(zhuǎn)化模式植物,以期對(duì)其功能進(jìn)行初步探索,為開發(fā)芍藥新品種和新異花色的分子育種提供可靠的理論依據(jù)和實(shí)踐意義。主要研究結(jié)果如下:(1)克隆了芍藥類黃酮途徑相關(guān)基因。提取芍藥'粉玉樓'花瓣總RNA,反轉(zhuǎn)錄合成cDNA第一鏈,根據(jù)課題組前期對(duì)相關(guān)基因的RACE結(jié)果,克隆獲得五個(gè)目的基因CHI、ANS、DFR、FLS、PAL,均包含完整編碼區(qū),推測(cè)氨基酸序列相似性與NCBI數(shù)據(jù)庫(kù)分別達(dá) 99%、98%、99%、100%、100%。(2)構(gòu)建了芍藥類黃酮途徑相關(guān)基因超表達(dá)載體。在擴(kuò)增獲得帶有合適的酶切位點(diǎn)的編碼區(qū)后,用SmaⅠ和Sac Ⅰ雙酶切目的基因CHI、ANS、DFR、PAL和表達(dá)載體質(zhì)粒pCAMBIA1301,用BamH Ⅰ和KpnⅠ雙酶切目的基因FLS和表達(dá)載體質(zhì)粒pCAMBIA1301,回收、連接目的片段與載體,分別成功構(gòu)建植物超表達(dá)載體pB1301-CHI、pB1301-ANS、pB1301-DFR、pB1301-FLS、pB1301-PAL共5個(gè)。測(cè)序驗(yàn)證后,采用凍融法將重組質(zhì)粒以及空載體轉(zhuǎn)化農(nóng)桿菌EHA105,獲得了用于植物轉(zhuǎn)化的材料。(3)獲得純合轉(zhuǎn)CHI、ANS、DFR、FLS、PAL基因擬南芥。利用農(nóng)桿菌介導(dǎo)Floral-dip法將目的基因CHI、ANS、DFR、FLS、PAL和空載體導(dǎo)入擬南芥,經(jīng)過T1代抗性篩選、GUS組織化學(xué)染色、T2和T3代分離比篩選,最終獲得轉(zhuǎn)CHI基因純合株系6個(gè),轉(zhuǎn)ANS基因純合株系2個(gè),轉(zhuǎn)DFR基因純合株系6個(gè),轉(zhuǎn)FLS基因純合株系3個(gè),轉(zhuǎn)PAL基因純合株系5個(gè),以及純合的轉(zhuǎn)空載株系2個(gè)。GUS染色、PCR鑒定及qRT-PCR分析顯示,純合轉(zhuǎn)基因擬南芥均能在基因組中檢測(cè)到目的基因,相對(duì)于對(duì)照組在轉(zhuǎn)錄水平表達(dá)水平明顯提高。(4)純合轉(zhuǎn)基因擬南芥表型初步分析。對(duì)轉(zhuǎn)基因株系的初步觀察發(fā)現(xiàn),轉(zhuǎn)CHI、FLS及PAL基因擬南芥與對(duì)照組相比,均無顯著差異。轉(zhuǎn)DFR和ANS基因的株系,雖然花器官與對(duì)照組相比無明顯差異,但在抽薹期,轉(zhuǎn)ANS基因株系部分蓮座葉葉背斑塊狀發(fā)紅,主脈基部略微發(fā)紅。轉(zhuǎn)DFR基因株系蓮座葉均葉色較深,部分葉片葉背發(fā)紅,DFR表達(dá)量最高的株系2葉背為深紫紅色。對(duì)轉(zhuǎn)基因各株系總黃酮與花色苷含量的測(cè)定有待進(jìn)一步研究。(5)獲得轉(zhuǎn)DFR、FLS基因煙草植株。利用農(nóng)桿菌介導(dǎo)葉盤法將目的基因DFR、FLS和空載體導(dǎo)入煙草,通過抗性篩選,獲得轉(zhuǎn)DFR基因植株43株,轉(zhuǎn)FLS基因植株66株,轉(zhuǎn)空載對(duì)照5株。通過GUS組織化學(xué)染色、PCR鑒定,隨機(jī)挑選的10株轉(zhuǎn)DFR基因煙草中陽性植株為8株,12株轉(zhuǎn)FLS基因煙草中陽性植株為7株,可認(rèn)為目的基因已整合進(jìn)入煙草基因組。通過qRT-PCR對(duì)陽性煙草中目的基因的轉(zhuǎn)錄水平進(jìn)行分析,結(jié)果顯示,與對(duì)照組相比,轉(zhuǎn)基因煙草中目的基因表達(dá)量明顯上升,表明目的基因均能在轉(zhuǎn)基因植株中正常表達(dá),且有一定促進(jìn)作用。由于時(shí)間及溫度原因,目前煙草還沒有抽薹開花,其生長(zhǎng)狀況及生物學(xué)特性上還未發(fā)現(xiàn)明顯差異,對(duì)其花色和花色苷的含量的變化有待進(jìn)一步實(shí)驗(yàn)。
[Abstract]:The flower color of the flower plant directly influences the ornamental value of the flower, and is the focus of the flower breeding. Paeonia lactifla is a kind of ornamental plant with considerable commodity value, but its color is mainly concentrated in the pink, red, purple, white and yellow lines, which is limited to its development to a certain extent. The improvement and innovation of the Chinese medicine flower color has been paid attention to, the main report focuses on the cross breeding, the research on the selection and breeding of the flower color by using the genetic engineering means is limited, At the same time, the regulation mechanism of the related gene has no profound knowledge. Many of the key genes in the flavonoid synthesis pathway, which are closely related to the suit, have been cloned and studied in many plants. after the PAL gene is a key gene for regulating and controlling the color and color in the synthesis route of the peony flavonoid, the target gene is cloned and the expression vector of the five genes is constructed, and the agrobacterium is used as the mediated transformation mode plant, so as to carry out preliminary exploration on the function thereof, and provides a reliable theoretical basis and practical significance for the development of the new variety of the peony and the molecular breeding of the new heterotype. The main results of this study were as follows: (1) The related genes of paeonol pathway were cloned. extraction of Paeonia lactiflora "powder jade building 'The first strand of the cDNA was synthesized by reverse transcription of the total RNA and reverse transcription. Five target genes, CHI, ANS, DFR, FLS, and PAL, were obtained according to the RACE results of the related genes in the earlier stage of the research group. The results showed that the similarity of the amino acid sequence and the NCBI database were 99%, 98% and 99%, respectively. 100%, 100%. and (2) constructing the hyperexpression vector of the peony flavonoid pathway related gene. after the coding region with the appropriate enzyme cutting site is obtained, the target gene CHI, ANS, DFR, PAL and the expression vector plasmid pCAMBIA1301 are cut with SmaI and Sac I double-enzyme digestion target genes CHI, ANS, DFR, PAL and expression vector pCAMBIA1301, The plant superexpression vectors pB1301-CHI, pB1301-ANS, pB1301-DFR, pB1301-FLS and pB1301-PAL were successfully constructed. After the sequencing and verification, the recombinant plasmid and the empty vector were transformed into Agrobacterium EHA105 by a freeze-thaw method, and a material for plant transformation was obtained. (3) obtaining pure-in-rotation CHI, ANS, DFR, FLS and PAL gene Arabidopsis. The target genes CHI, ANS, DFR, FLS, PAL and empty vector were introduced into arabidopsis by Agrobacterium-mediated Flourral-dip method, and then were selected by T1 generation resistance screening, GUS histochemical staining, T2 and T3 generation. 6 of the pure strain of the transgenic DFR gene, 3 of the pure strain of the transgenic FLS gene, 5 of the pure strain of the transgenic PAL gene, and 2 of the pure-in-one transgenic no-load strains. GUS staining, PCR identification and qRT-PCR analysis showed that both the pure and transgenic Arabidopsis can detect the target gene in the genome, and the expression level of the transcription level in the control group is obviously improved. (4) Preliminary analysis of the phenotype of pure-in-transgenic Arabidopsis. The results of the preliminary observation on the transgenic lines showed no significant difference in the expression of CHI, FLS and PAL and the control group. The lines of the DFR and ANS genes, although there was no significant difference between the flower organ and the control group, in the pumping stage, the leaf of the leaf of the lotus leaf of the ANS gene line was red, and the base of the main vein was slightly reddish. The leaf color of the leaf of the RFR gene was deep, the leaves of the leaf were red, and the 2-leaf back of the strain with the highest degree of DFR expression was deep-purplish. The determination of the content of total flavone and flower color of all the transgenic lines is to be further studied. (5) obtaining the transformed DFR and FLS gene tobacco plants. The target gene DFR, FLS and the empty vector were introduced into the tobacco by Agrobacterium-mediated leaf disc method, and the transgenic plants of the transgenic DFR gene were obtained by resistance screening, and 66 strains of the transgenic FLS gene and 5 strains of no-load control were obtained. By means of GUS histochemical staining and PCR identification, 8 of the 10 transgenic DFR gene plants selected randomly, and 7 of the 12 transgenic FLS genes, can be considered to be integrated into the tobacco genome. Through the analysis of the transcription level of the target gene in the positive tobacco by qRT-PCR, the results showed that the expression of the target gene in the transgenic tobacco obviously increased compared with the control group, indicating that the target gene can be normally expressed in the transgenic plant and has a certain promoting effect. Because of the time and temperature, there is no significant difference in the growth and biological characteristics of the tobacco, and the change of the content of the color and the flower color of the tobacco is to be further tested.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S682.12
[Abstract]:The flower color of the flower plant directly influences the ornamental value of the flower, and is the focus of the flower breeding. Paeonia lactifla is a kind of ornamental plant with considerable commodity value, but its color is mainly concentrated in the pink, red, purple, white and yellow lines, which is limited to its development to a certain extent. The improvement and innovation of the Chinese medicine flower color has been paid attention to, the main report focuses on the cross breeding, the research on the selection and breeding of the flower color by using the genetic engineering means is limited, At the same time, the regulation mechanism of the related gene has no profound knowledge. Many of the key genes in the flavonoid synthesis pathway, which are closely related to the suit, have been cloned and studied in many plants. after the PAL gene is a key gene for regulating and controlling the color and color in the synthesis route of the peony flavonoid, the target gene is cloned and the expression vector of the five genes is constructed, and the agrobacterium is used as the mediated transformation mode plant, so as to carry out preliminary exploration on the function thereof, and provides a reliable theoretical basis and practical significance for the development of the new variety of the peony and the molecular breeding of the new heterotype. The main results of this study were as follows: (1) The related genes of paeonol pathway were cloned. extraction of Paeonia lactiflora "powder jade building 'The first strand of the cDNA was synthesized by reverse transcription of the total RNA and reverse transcription. Five target genes, CHI, ANS, DFR, FLS, and PAL, were obtained according to the RACE results of the related genes in the earlier stage of the research group. The results showed that the similarity of the amino acid sequence and the NCBI database were 99%, 98% and 99%, respectively. 100%, 100%. and (2) constructing the hyperexpression vector of the peony flavonoid pathway related gene. after the coding region with the appropriate enzyme cutting site is obtained, the target gene CHI, ANS, DFR, PAL and the expression vector plasmid pCAMBIA1301 are cut with SmaI and Sac I double-enzyme digestion target genes CHI, ANS, DFR, PAL and expression vector pCAMBIA1301, The plant superexpression vectors pB1301-CHI, pB1301-ANS, pB1301-DFR, pB1301-FLS and pB1301-PAL were successfully constructed. After the sequencing and verification, the recombinant plasmid and the empty vector were transformed into Agrobacterium EHA105 by a freeze-thaw method, and a material for plant transformation was obtained. (3) obtaining pure-in-rotation CHI, ANS, DFR, FLS and PAL gene Arabidopsis. The target genes CHI, ANS, DFR, FLS, PAL and empty vector were introduced into arabidopsis by Agrobacterium-mediated Flourral-dip method, and then were selected by T1 generation resistance screening, GUS histochemical staining, T2 and T3 generation. 6 of the pure strain of the transgenic DFR gene, 3 of the pure strain of the transgenic FLS gene, 5 of the pure strain of the transgenic PAL gene, and 2 of the pure-in-one transgenic no-load strains. GUS staining, PCR identification and qRT-PCR analysis showed that both the pure and transgenic Arabidopsis can detect the target gene in the genome, and the expression level of the transcription level in the control group is obviously improved. (4) Preliminary analysis of the phenotype of pure-in-transgenic Arabidopsis. The results of the preliminary observation on the transgenic lines showed no significant difference in the expression of CHI, FLS and PAL and the control group. The lines of the DFR and ANS genes, although there was no significant difference between the flower organ and the control group, in the pumping stage, the leaf of the leaf of the lotus leaf of the ANS gene line was red, and the base of the main vein was slightly reddish. The leaf color of the leaf of the RFR gene was deep, the leaves of the leaf were red, and the 2-leaf back of the strain with the highest degree of DFR expression was deep-purplish. The determination of the content of total flavone and flower color of all the transgenic lines is to be further studied. (5) obtaining the transformed DFR and FLS gene tobacco plants. The target gene DFR, FLS and the empty vector were introduced into the tobacco by Agrobacterium-mediated leaf disc method, and the transgenic plants of the transgenic DFR gene were obtained by resistance screening, and 66 strains of the transgenic FLS gene and 5 strains of no-load control were obtained. By means of GUS histochemical staining and PCR identification, 8 of the 10 transgenic DFR gene plants selected randomly, and 7 of the 12 transgenic FLS genes, can be considered to be integrated into the tobacco genome. Through the analysis of the transcription level of the target gene in the positive tobacco by qRT-PCR, the results showed that the expression of the target gene in the transgenic tobacco obviously increased compared with the control group, indicating that the target gene can be normally expressed in the transgenic plant and has a certain promoting effect. Because of the time and temperature, there is no significant difference in the growth and biological characteristics of the tobacco, and the change of the content of the color and the flower color of the tobacco is to be further tested.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S682.12
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