类黄酮合成关键酶在苔藓适应极地强紫外辐射中的作用

发布时间：2018-06-17 20:12

本文选题：南极苔藓 + 紫外辐射　；参考：《山东大学》2017年硕士论文

【摘要】：南极洲位于地球南端,四周被三大洋包围,与其他大陆隔离,大陆面积约1240万平方千米,占地球陆地总面积的十分之一,但仅分布850多种植物,且绝大部分为低等植物。强紫外是制约南极陆生植物生长发育的主要因素之一。苔藓植物(Bryophytes)是南极大陆分布最广、数量最多的绿色植物。苔藓适应极端环境强辐射的机制与其特殊的结构基因及代谢途径密切相关。类黄酮化合物(Flavonoids)是植物体内最大的一类多羟基酚类次生代谢产物,从高等植物到较低等的植物类群都有分布,在植物适应紫外辐射过程中起着重要作用。在高等植物中,类黄酮代谢途径较为清晰,其起始于苯丙烷类代谢途径,经查尔酮合酶和查尔酮异构酶催化生成类黄酮化合物的前体物质柚皮素(黄烷酮);柚皮素作为类黄酮代谢途径的前体化合物,被黄烷酮3-羟化酶、黄酮醇合成酶、花青素合成酶和黄酮合酶等酶催化进入不同的分支代谢途径。然而低等植物的类黄酮代谢途径仍不清楚,目前仅有小立碗藓查尔酮合酶家族及查尔酮异构酶,和一个苔类的黄酮合酶的报道。本论文分析了紫外辐射下,南极丝瓜藓类黄酮化合物的代谢变化;克隆了类黄酮代谢途径的关键酶查尔酮合酶家族基因和一个黄酮合酶基因(Flavone synthase,FNS),分析了其对非生物胁迫的响应特征,研究了FNS重组蛋白的体外的催化活性和在拟南芥中的体内催化活性。具体结果如下:1南极苔藓紫外辐照条件下黄酮类物质的响应利用UV-B辐射处理南极苔藓(丝瓜藓属)和常温苔藓(丝瓜藓属,来自山东省),经UV-B辐射处理100 h后,结果发现常温苔藓茎叶体上端枯死,当在黑暗条件下恢复12 h后,枯死程度继续加剧;而南极苔藓的拟茎由嫩绿色变为深褐色,恢复生长后拟茎变为浅棕色。初步表明,与常温苔藓比,南极苔藓对UV-B辐射的抗性更强,这种抗性可能与多酚类化合物有关。利用HPLC研究了 UV-B辐射处理对南极丝瓜藓类黄酮化合物代谢谱的影响。结果发现,UV-B处理不仅增加了类黄酮化合物的含量,还诱导新的化合物的合成。2南极苔藓查尔酮合酶家族基因的特征分析查尔酮合酶(Chalcone synthase,CHS)是植物类黄酮生物合成途径中的第一个关键酶,在植物抵御外界环境胁迫中发挥重要作用。利用RACE技术从南极丝瓜藓cDNA中获取了 6条开放阅读框约1200bp大小片段的查尔酮合酶家族基因(Pn021、Pn444、Pn768、Pn088、Pn847 和 Pn270)。序列比对分析发现,PnCHS与来自其它物种的查尔酮合酶的蛋白序列相似度在24.0%-84.0%之间;Pn444、Pn088和Pn768具有保守的Cys164、His303和Asn336(氨基酸序号以苜蓿蛋白标出)催化三联体及GFGPG基序和两个保守的与底物结合的口袋。利用近邻比对法构建PnCHS的系统进化树,结果显示苔藓植物的CHS聚类到同一分枝,位于菌类和高等植物的中间。构建35S::Pn444::GFP/pBI221瞬时表达载体,利用PEG介导法转化拟南芥原生质体,荧光共聚焦显微镜下观察绿色荧光蛋白信号,发现目的蛋白主要分布于细胞内膜性细胞器。利用qRT-PCR技术分析了不同胁迫处理下,PnCHS家族基因的响应情况。结果表明,UV-B辐射下,PnCHS家族基因的表达水平均上调表达;低温处理下PnCHS表达水平都下调表达;高盐处理后南极苔藓查尔酮合酶基因Pn021、Pn088和Pn444的表达水平下调,而Pn270和Pn847的表达水平上调;干旱胁迫后,Pn021、Pn444和Pn847的表达水平显著上调;MeJA和ABA处理后,Pn444和Pn847的表达水平显著增加。3南极苔藓黄酮合酶的体内外酶活功能分析黄酮合酶(Flavone synthase)是类黄酮代谢途径中重要的双加氧酶之一,催化黄烷酮在C环2-3位形成双键。通过筛选南极苔藓转录组数据库,克隆得到一个和黄酮合酶同源性较高的基因(PnFNS),与其他20DD家族相似性为20.0-80.0%,包含HXDX～50HK～10RXS motif、Fe2+结合位点、2-酮戊二酸结合位点以及柚皮素催化位点。通过构建系统进化树,我们发现PnFNS与小立碗藓中的相应同源蛋白类聚在同一分枝;以拟南芥花青素合酶(2brt.pdb)晶体结构为模板,利用SWISS-MODEL在线对PnFNS蛋白进行同源建模,预测其催化机制。通过构建荧光蛋白融合载体35S::PnFNS::GFP/pBI221转入拟南芥原生质体,观察显示荧光蛋白位于膜性细胞器,表明PnFNS可能主要定位在细胞膜和内质网。构建PnFNS-pET28a进行异源表达,体外实验发现,重组蛋白仅能催化柚皮素生成芹菜素,对二氢槲皮素、芹菜素、山奈酚、槲皮素均不发生反应。通过pH3.0-11.0的缓冲液分析PnFNS的最适pH,结果发现,PnFNS在低于pH4.0的反应液内催化活性很低,pH4.0-7.0之间酶活性随着pH的升高而增加,大于pH7.0后酶活性又迅速下降。以pH7.0为最适pH进行梯度温度反应,发现PnFNS对低温不敏感,在0-20℃期间均有较高活性;高于30℃时,酶活性迅速下降。在pH7.0和15℃条件下,研究了不同离子对PnFNS的催化活性;结果表明,Mg2+、Ca2+、Mn2+和Cu2+对酶活性有提升作用,Co2+对酶活性有部分抑制作用,添加离子螯合剂Na2EDTA后完全抑制了酶活性,表明PnFNS是金属离子依赖型低温酶。将3 mg的野生型拟南芥和过表达PnFNS的转基因拟南芥(AtOE)种子放入加有0或200 μM的柚皮素的液体1/2MS中静止培养10d。结果发现,正常培养情况下,野生型和转基因拟南芥的生长没有差别,但是含有200 μM柚皮素的1/2MS中,过表达PnFNS在很大程度上解除了柚皮素对植株生长的抑制,转基因拟南芥的长势显著强于野生型拟南芥。利用HPLC分析植物类黄酮代谢谱,发现柚皮素在野生型和转基因拟南芥中都没有明显的积累,但是野生型的拟南芥中积累一个保留时间早于柚皮素的物质(推测为:6-丙酰基-α-D-葡萄糖基-(1→7)-橙皮素),而转基因拟南芥中积累了较多的芹菜素。表明南极苔藓PnFNS在拟南芥体内具有和体外类似的催化反应。将3周龄的野生型拟南芥和过表达PnFNS的拟南芥在添加UV-B(20 μW/um2)的全波长光源下处理6h,然后恢复生长2d,观察叶片损伤情况并通过DAB染色检测叶片活性氧的含量。结果发现,过表达拟南芥叶片坏死面积和活性氧水平均明显少于野生型,表明过表达PnFNS拟南芥增强了植物对紫外的耐受能力。
[Abstract]:Antarctica is located at the southern end of the earth, surrounded by three oceans and isolated from other continents. The continent is about 12 million 400 thousand square kilometers, accounting for 1/10 of the total area of the earth's land, but only 850 kinds of plants are distributed, and most of them are low plants. The strong ultraviolet (Bryophy) is one of the main factors restricting the growth and development of Antarctic Lu Shengzhi. Bryophytes (bryophyte) TES) is the most widely distributed and largest green plant in the Antarctic continent. The mechanism of moss adapted to extreme environmental strong radiation is closely related to its special structural genes and metabolic pathways. The flavonoid compound (Flavonoids) is the largest secondary metabolite of polyhydroxyphenols in plants, from higher plants to lower plant groups. Distribution, plays an important role in the process of plant adaptation to ultraviolet radiation. In higher plants, the metabolic pathway of flavonoids is clearer. It begins in the phenylpropanoid pathway and catalyzes the production of the precursor of the flavonoids by chalcone synthase and chalcone isomerase in the precursor of naringin (Huang Wantong); naringenin is a precursor to the metabolic pathway of flavonoids. Body compounds, which are catalyzed by flavanone 3- hydroxylase, Flavonol Synthase, anthocyanin synthetase, and flavonoid synthase, are catalyzed into different branch metabolic pathways. However, the metabolic pathways of flavonoids in lower plants are still unclear. Currently, only the chalcone synthase family and the chalcone isomerase, and the flavonoid synthase of a liverwort are reported in the lower plants. In this paper, the metabolic changes of flavonoids in the Antarctic gourd moss were analyzed under ultraviolet radiation, and the key enzymes of the flavonoid metabolism pathway, chalcone synthase family gene and a flavonoid synthase gene (Flavone synthase, FNS), were cloned, and their response to abiotic stress was analyzed, and the catalytic activity of FNS recombinant protein in vitro was studied. The specific results are as follows: 1 the response of the flavonoids in the UV irradiated Antarctic moss under the UV irradiation conditions by UV-B radiation treatment of the moss of Antarctic (MOSS MOSS) and the moss of normal temperature (from Shandong province) and after 100 h treatment by UV-B radiation, the fruit found that the upper end of the stem and leaf of the moss at normal temperature was dead, when in the dark strip. After the recovery of 12 h, the death degree continued to intensify, and the pseudo stem of the Antarctic moss changed from tender green to dark brown, and the pseudo stem became light brown after the recovery. It was preliminarily indicated that the resistance of Antarctic moss to UV-B radiation was stronger than that of moss at normal temperature. This resistance may be related to polyphenols. The use of HPLC to study the treatment of UV-B radiation to Antarctica. The results showed that the UV-B treatment not only increased the content of flavonoids, but also induced the synthesis of new compounds,.2, the characteristics of the gene of chalcone synthase in the moss Antarctica, the Chalcone synthase (CHS) was the first key enzyme in the biosynthesis pathway of the plant flavonoids. The RACE technique was used to obtain 6 chalcone synthase family genes (Pn021, Pn444, Pn768, Pn088, Pn847 and Pn270) from the Antarctic gourd moss cDNA by using RACE technology. The sequence alignment analysis found that PnCHS and the protein of chalcone synthase from other species Sequence similarity is between 24.0%-84.0%; Pn444, Pn088 and Pn768 have the conservative Cys164, His303 and Asn336 (amino acid sequence number with alfalfa protein) to catalyze the three body and GFGPG motif and two conservative pockets with the substrate. The phylogenetic tree of PnCHS is constructed by near neighbor comparison method. The results show that the CHS cluster of the moss plants to the same one. Branches, located in the middle of fungi and higher plants. Construct 35S:: Pn444:: GFP/pBI221 transient expression vector, transform Arabidopsis Protoplast by PEG mediated method, observe green fluorescent protein signal under fluorescence confocal microscope, find that the target protein is mainly distributed in cell endocardium cell organelles. The different stress treatment is analyzed by qRT-PCR technology. The response of PnCHS family genes showed that the expression level of PnCHS gene expression was up regulated under UV-B radiation; PnCHS expression level was down regulated under low temperature treatment; the expression level of Pn021, Pn088 and Pn444 in Antarctic lichen chalcone synthase gene was down regulated, and the expression level of Pn270 and Pn847 was up-regulated after high salt treatment; drought stress was up. After the treatment of Pn021, Pn444 and Pn847, the expression level of MeJA and ABA significantly increased the expression level of Pn444 and Pn847 in.3 Antarctic moss flavonoid synthase activity analysis of the flavonoid synthase (Flavone synthase), one of the most important bioxygenase in the flavonoid metabolic pathway, catalyzing the formation of a double bond in the 2-3 position of the flavanone in the C ring. Screening the database of the Antarctic moss transcriptional group to clone a gene (PnFNS) with a high homology of the flavonoid synthase, which is similar to that of other 20DD families, including HXDX to 50HK to 10RXS motif, Fe2+ binding site, 2- ketone glutaric acid binding site and naringin catalytic site. By constructing phylogenetic tree, we found PnFNS and small. The corresponding homologous proteins in the erect moss were clustered in the same branch; using the Arabidopsis anthocyanin synthase (2brt.pdb) crystal structure as a template, the homologous modeling of PnFNS protein was established by SWISS-MODEL online, and its catalytic mechanism was predicted. The fluorescent protein fusion carrier, 35S:: PnFNS:: GFP/pBI221, was transferred into the Arabidopsis protoplast, and the fluorescent eggs were observed and displayed. It is located in the membranous organelle, indicating that PnFNS may be mainly located in the cell membrane and endoplasmic reticulum. PnFNS-pET28a is constructed for the heterologous expression. In vitro, the recombinant protein can only catalyze the production of naringin, and it does not respond to two hydrogen quercetin, apigenin, kaempferol and quercetin, and the optimum analysis of PnFNS by pH3.0-11.0 buffer solution. PH, the results showed that the catalytic activity of PnFNS in the reaction liquid below pH4.0 was very low, the activity of pH4.0-7.0 increased with the increase of pH, and the activity of the enzyme decreased rapidly after the pH7.0. PH7.0 was the optimum pH for the gradient temperature reaction. It was found that PnFNS was insensitive to low temperature and had high activity at 0-20 degrees C, and the enzyme activity was rapid when higher than 30. Down. The catalytic activity of different ions on PnFNS was studied at pH7.0 and 15 C. The results showed that Mg2+, Ca2+, Mn2+ and Cu2+ had a promotion of enzyme activity, Co2+ had partial inhibition on the activity of enzyme, and the activity of the enzyme was completely suppressed after the addition of the ion chelating agent Na2EDTA. It was indicated that PnFNS was a metal ion dependent cryogenic enzyme. The wild type of 3 mg was found. In Arabidopsis and PnFNS transgenic Arabidopsis (AtOE) seeds in the liquid 1/2MS with 0 or 200 mu M in the liquid 1/2MS, it was found that there was no difference in the growth of wild type and transgenic Arabidopsis in normal culture, but the overexpression of PnFNS in 1/ 2MS containing 200 u M naringin greatly relieved the grapefruit. The growth of transgenic Arabidopsis was significantly stronger than that of wild Arabidopsis. Using HPLC to analyze the metabolic profiles of flavonoids in plants, it was found that naringin had no obvious accumulation in wild type and transgenic Arabidopsis, but in wild Arabidopsis, the accumulation time was earlier than that of naringenin (presumably 6-). The propanoyl - alpha -D- glucosamine - (1 - 7) - hesperidin, and the accumulation of more apigenin in transgenic Arabidopsis. It shows that the Antarctic moss PnFNS has a similar catalytic reaction in the body with the Arabidopsis in vitro. The 3 week old wild Arabidopsis and the Arabidopsis thaliana overexpressing PnFNS are treated with the full wavelength light source of UV-B (20 mu) with the full wavelength light source, and then the Arabidopsis thaliana is treated with the whole wavelength light source. The growth of 2D was restored and the leaf damage was observed and the content of active oxygen in leaves was detected by DAB staining. The results showed that over expression of the leaf necrosis area and active oxygen water of Arabidopsis thaliana was significantly less than that of the wild type, indicating that over expression of PnFNS Arabidopsis enhanced the tolerance of plants to ultraviolet.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

【参考文献】