梨（Pyrus）果实石细胞的结构成分分析及相关酶基因的克隆
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摘要
石细胞是影响梨果实品质的重要因素之一。前人研究已明确遗传背景是石细胞发育的内在因素,而生态条件及栽培管理措施同样可以影响梨果实石细胞的发育。但目前的研究主要在石细胞含量、大小、分布等对梨果实品质影响的认识上。此外,在生理学角度也对石细胞发育过程进行了研究,但是在研究过程中对石细胞由大量木质素构成这一重要的化学本质研究较少。本研究以石细胞较多、肉质粗糙的‘砀山酥梨’(Pyrus bretschneideri cv.Dangshansuli)为试验材料,同时用石细胞较少、肉质细腻的‘幸水’(Pyrus pyrifolia cv.Kousui)作为品种对照,系统地开展了对梨果实石细胞结构和成分的研究,并对梨果实中PAL(苯丙氨酸解氨酶)、CAD(肉桂醇脱氢酶)、POD(过氧化物酶)基因进行了克隆和序列分析,主要结果如下：
一、以肉质较粗糙的‘砀山酥梨’为试验材料,并用肉质较细腻的‘幸水’为对照,利用Wiesner试剂,高锰酸钾染色及木质素自发荧光的特性,结合溴乙酰化学分析法、普通光学显微镜、荧光显微镜、激光共聚焦显微镜、透射电子显微镜、扫描电子显微镜、电感耦合等离子体发射光谱仪、X射线能谱仪及图形分析软件研究了梨果实中石细胞的超微结构和木质素的分布特性。‘砀山酥梨’和‘幸水’果实石细胞中木质素含量分别达到了29.83%和24.55%。果肉中的蓝色自发荧光主要来自于石细胞,并且‘砀山酥梨’的荧光能量值稍高于‘幸水’。石细胞中木质素由细胞外层发生,逐步向质膜发展,分层沉积,直至形成充满细胞腔的次生壁,并伴生有典型的单纹孔结构。在木质化过程中,石细胞的整个细胞壁明显分为木质化程度不同的四层结构,即复中层(CML)、次生壁1(S1)、次生壁2(S2)及高度木质化的、具有典型应压木特征的外层次生壁2(S2L)。
二、以‘砀山酥梨’为试验材料,并以‘幸水’为对照,提取纯化了梨果实石细胞中的木质素,并通过谱学方法对其特性进行了研究。上述两种来源的木质素结构类型基本一致,但提取的‘幸水’木质素重均分子量(WMW),分子量分散系数(MWD)都要大于‘砀山酥梨’。相应地,其玻璃化温度(GTT)也要高于‘砀山酥梨’,为160℃。石细胞木质素的紫外光谱吸收表现出其典型的苯环结构,并在红外光谱上得到验证。综合Maule试剂染色、红外光谱和硝基苯氧化产物HPLC分析的结果：发现石细胞木质素含有大量的愈创木基单元和一部分紫丁香基结构单元,而对羟基苯丙烷结构单元很少。进一步结合对1H-NMR和13C-NMR波谱的分析,初步明确梨果实石细胞木质素为典型的双子叶植物木质素,但其愈创木基结构单元含量高于其他树种,愈创木基与紫丁香基结构单元比例(G／S)高达4,接近于裸子植物,表现出软木木质素的结构特性。
三、梨果实石细胞在幼果中分布密度最高,此后因果实生长和膨大的“稀释”作用,其分布密度逐渐降低。石细胞团的大小随果实的生长一直增大。苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)和多酚氧化酶(PPO)的活性变化规律基本一致,其活性高峰都出现在幼果发育期,后随着果实发育逐渐下降。套袋处理能够抑制‘砀山酥梨’果实石细胞的发育,而且套袋越早效果越显著,相应PAL、POD、PPO的活性也较低。通过对比不同时期套袋处理的不同发育阶段果实内酶活性的平均值与其相应的石细胞最终含量,发现梨果实石细胞最终含量与POD、PAL、PPO活性之间呈正相关关系,相关系数分别为：RPOD=0.8035; RPAL=0.8134; RPPO=0.9186。进一步对‘砀山酥梨’进行喷CaCl2和多效唑处理,并以‘幸水’为品种对照,发现在两个品种上钙处理均能降低果实中木质素、石细胞含量和PAL、POD、PPO的总活性,而多效唑则起着相反的作用。酶活性和木质素、石细胞含量之间成正相关关系,并且细胞壁结合过氧化物酶(PODⅡ)在石细胞发育过程中起更大的作用。
四、以‘砀山酥梨’为试验材料,研究了梨果实中过氧化物酶(POD)、过氧化氢(H2O2)与石细胞及木质素之间的关系。成熟‘砀山酥梨’果肉干重的36.03%为石细胞,15.23%为木质素,其中有70.6%的木质素存在于石细胞中,石细胞重量的29.83%为木质素。通过徒手切片对果实中石细胞及过氧化物酶进行组织染色定位,发现无论是在横切面还是纵切面上,两者的分布区域都保持一致。木质素在果实发育初期大量合成,而细胞质过氧化物酶(PODⅠ)和细胞壁结合过氧化物酶(PODⅡ)都随果实的发育而逐渐降低,但PODⅡ对木质素单体类似物丁香醛连氮(Syringaldazine)的催化能力要远远高于POD I. POD和H2O2在梨果实细胞中的分布主要集中在细胞壁和细胞间隙之中。进一步通过DEAE离子交换层析柱分离PODⅡ中的碱性和酸性PODⅡ,并进行底物特异性分析,发现PODⅡ对Syringaldazine的催化主要由碱性PODⅡ完成。在梨果实中极有可能是碱性的细胞壁结合过氧化物酶(PODⅡ)在过氧化氢的存在下参与了木质素合成的最后一步反应,促进了石细胞的形成。
五、提取了‘砀山酥梨’及‘幸水’果肉总RNA,并通过RT-PCR、克隆和测序,获得了苯丙氨酸解氨酶(PAL),过氧化物酶(POD)和肉桂醇脱氢酶(CAD)的cDNA片段。在核苷酸水平上,2个品种PAL基因片段大小为653bp,只有3个碱基的差异,编码1个完全相同的氨基酸序列,该序列由217个残基组成。所推导的氨基酸序列与西洋梨Pyrus communis (ABB70117)、甜樱桃Prunus avium (AAC78457)、树莓Rubus idaeus (AAF40224)和野生橘Citrus clementina×Citrus reticulata(CAB42793)氨基酸序列的相似性分别达到98.6%、98.6%97.7%和97.2%。此外,它们含有4个与其他PAL蛋白质相似的蛋白质保守活性位点。
两个品种CAD基因的cDNA片段大小为689bp,核苷酸序列同源性为99.4%,有4个碱基的差异,而氨基酸序列相似性为99.6%,只有1个氨基酸残基的差异。所推导的氨基酸序列与苹果Malus domestica (AAC06319)、梅Prunus mume (BAE48658)、枇杷Eriobotrya japonica (ABV44810)和葡萄Vitis vinifera(CA021890)氨基酸序列的相似性分别达到97.8%、95%、92.6%和83.4%。
两个品种POD基因的cDNA片段大小约为574bp,编码191个氨基酸残基,它们之间的核苷酸序列和氨基酸序列没有任何差异。推导的氨基酸序列与烟草Nicotiana tabacum (AAD33072)、文心兰Oncidium Gower Ramsey (ABC02343)、盐芥Thellungiella halophila (ABU54828)和拟南芥Arabidopsis thaliana (CAA6686)氨基酸序列的相似性分别为84.3%、86.4%、86.9%和86.4%。此外,它们含有与其他POD蛋白相似的3个保守活性位点和2个底物结合位点。
结果表明,本研究克隆到的PB-PAL(登录号：FJ478149). PP-PAL(登录号：FJ478150). PB-CAD(登录号：FJ478151). PP-CAD(登录号：FJ478152). PB-POD(登录号：FJ478153)和PP-POD(登录号：FJ478154)就是梨果实中的PAL、CAD和POD基因片段。
Sclereid or stone cell is one of the crucial factors impacting grit texture in pear fruit. It is reported that genetic background is the intrinsic factor for sclereids development; however the culture practices can also influence their development consumingly. Unfortunately, no great progress is achieved on pear sclereids research till now; researchers always omit lignin-the chemical nature of sclereids, and few papers are found focusing on pear sclereid lignin directly. In this study,'Dangshansuli'pear which contains high sclereid content was used as the major material, and'Kousui'which has low content of sclereid as accessorial material to characterize sclereid structure and composition; also, PAL (Phenylalanine Ammonia Lyase), CAD (Cinnamyl Alcohol Dehydrogenase) and POD (Peroxidase) genes were cloned and analysed. The results are as follows:
1. Anatomy, ultrastructure and lignin distribution in stone cells from the fruit of two Pyrus species (Pyrus bretschneideri cv. Dangshansuli and Pyrus pyrifolia cv. Kousui) were examined by using light microscopy (LM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray energy spectrometer and inductive coupled plasma emission spectrometer (ICP) as well as autofluorescence analysis. Sections stained with phloroglucinol-HCl revealed the presence of lignin in stone cells, and showed that stone cells were distributed in a mosaic pattern in the flesh, with larger stone cells concentrated around the core and smaller ones in the pericarp. There were no obvious differences in stone cell structure between the two varieties, but stone cell size and content was much greater in'Dangshansuli'than in'Kousui.'Further, lignin accounted for 29.8% of stone cell composition in'Dangshansuli', a significantly higher proportion than that in 'Kousui'(24.6%), basing on acetyl bromide method; this result was confirmed by autofluorescence analysis. More detailed information on lignin distribution across the cell wall was obtained by TEM combined with the KMnO4 staining technique. In TEM images, cell walls of both pear varieties were typically divided into four layers:compound middle lamella (CML), secondary wall 1 (S1), outer secondary wall 2 (S2L) and secondary wall 2 (S2), with different staining intensities for different lignin concentrations in those regions.
2. Lignin isolated from sclereids of'Dangshansuli'and'Kousui'pear were studied with several spectroscopies, such as ultra violet spectroscopy (UV), fourier transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC),1H nuclear magnetic resonance (1H-NMR) and 13C nuclear magnetic resonance (13C-NMR). The lignin were the same in structural information, however the weight-average molecular weight (WMW) and molecular weight distribution (MWD) of'Kousui'lignin both were higher than 'Dangshansuli'lignin, consequently, it had higher glass transition temperature (GTT). Typical benzene structure showed in UV spectra was also confirmed by IR spectra. Great vanillin and some syringaldehyde found in lignin nitrobenzene oxidation products and Maule reagent staining result indicated the presence of guaiacyl unit in the lignin. Further, with the aids of 1H and 13C-NMR spectra, lignin in pear sclereids was characterized as typical guaiacyl-syringyl lignin (G-S lingnin), however it has higher content guaiacyl unit than other angiosperm, the ratio for guaiacyl and syringyl unit was as high as 4, more closely to gymnosperm.
3. Distribution density of sclereids was highest in pear fruitlet and then descended with fruit development; however the size of sclereid cluster increased gradually till fruit mature. With regards to phenylalanine ammonia lyase (PAL), peroxidase (POD) and polyphenol oxidase (PPO), their activity peaks always appeard in fruitlets, and then declined gradually in the course of fruit development. Fruit bagging had good effects on'Dangshansuli'fruit quality; it can reduce the content and size of sclereid in mature fruit; furthermore, bagging stage had influence on the effects, the earlier the better. Also, CaCl2 showed the same effects as fruit bagging on both'Dangshansuli'and'Kousui', while PP333 had the opposite result. All the data from this section showed a positive relationship between the enzymes' activities and lignin, sclereid content and size. Especially the cell wall bounded peroxidase (PODⅡ) played more important role in cell lignification.
4. The relationship among peroxidase, hydrogen peroxide, lignin and sclereid was studied by using'Dangshansuli'as material. Sclereids accounted for a big amount in dry fruit weight as 36.03%, and they contained 29.83%lignin which accounted for 70.6%of the total in fruit. Peroxidase activity was found almost around sclereids in free hand sections, and in the cell wall at cellular level. Besides, hydrogen peroxide was also localized at the similar regions, such as cell wall, cell corner and intercellular layer. Lignin was synthesized in big scale along with a burst of hydrogen peroxide in young fruit; also the soluble peroxidase (PODⅠ) and cell wall bounded peroxidase (PODⅡ) showed the same changing tendency, however the PODⅡalways possessed higher affinity to syringaldazine-an analog of lignin monomer. Further study basing on substrate affinity analysis demonstrated that it was the basic PODⅡshowing high catalytic efficiency. Lignin biosynthesis and sclereid development were related to basic PODⅡ.
5. Two PAL genes, two POD genes, and two CAD genes were cloned and sequenced using RT-PCR from'Dangshansuli'and'Kousui'pear respectively. In nucleotide sequence, the two PAL genes had 3 bp differences, and coded an identical amino acid sequence which comprised of 217 residues. Meanwhile, the amino acid sequence of the two PAL was 98.6%identical to PAL from Pyrus communis (ABB70117),98.6% identical to PAL from Prunus avium (AAC78457),97.7%identical to PAL from Rubus idaeus (AAF40224), and 97.2%indentical to PAL from Citrus clementina×Citrus reticulata (CAB42793) respectivity. Further, they were found to have 4 similar active sites with PAL from other plant species.
The two CAD genes had 4 bp differences resuting in one residue differences. The deduced amino acid sequence of the two CAD was 97.8%identical to CAD from Malus domestica (AAC06319),95%identical to CAD from Prunus mume (BAE48658), 92.6%identical to CAD from Eriobotrya japonica (ABV44810) and 83.4%identical to CAD from Vitis vinifera (CAO21890) respectively.
The two POD genes were identical in both nucleotide sequences and deduced amino acid sequences. Deduced amino acid sequence of the POD from pear was 84.3% identical to POD from Nicotiana tabacum (AAD33072),86.4%identical to POD from Oncidium Gower Ramsey (ABC02343),86.9%identical to POD from Thellungiella halophila (ABU54828) and 86.4%identical to POD from Arabidopsis thaliana (CAA6686) respectively. Moreover, POD from these two pears was found to have 3 similar active sites and 2 substrate bounding sites with POD from other plant species.
All the results from above showed PB-PAL (Accession No. FJ478149), PP-PAL (Accession No. FJ478150), PB-CAD (Accession No. FJ478151), PP-CAD (Accession No. FJ478152), PB-POD (Accession No. FJ478153) and PP-POD (Accession No. FJ478154) were the PAL, CAD and POD genes in pear fruit.
一、以肉质较粗糙的‘砀山酥梨’为试验材料,并用肉质较细腻的‘幸水’为对照,利用Wiesner试剂,高锰酸钾染色及木质素自发荧光的特性,结合溴乙酰化学分析法、普通光学显微镜、荧光显微镜、激光共聚焦显微镜、透射电子显微镜、扫描电子显微镜、电感耦合等离子体发射光谱仪、X射线能谱仪及图形分析软件研究了梨果实中石细胞的超微结构和木质素的分布特性。‘砀山酥梨’和‘幸水’果实石细胞中木质素含量分别达到了29.83%和24.55%。果肉中的蓝色自发荧光主要来自于石细胞,并且‘砀山酥梨’的荧光能量值稍高于‘幸水’。石细胞中木质素由细胞外层发生,逐步向质膜发展,分层沉积,直至形成充满细胞腔的次生壁,并伴生有典型的单纹孔结构。在木质化过程中,石细胞的整个细胞壁明显分为木质化程度不同的四层结构,即复中层(CML)、次生壁1(S1)、次生壁2(S2)及高度木质化的、具有典型应压木特征的外层次生壁2(S2L)。
二、以‘砀山酥梨’为试验材料,并以‘幸水’为对照,提取纯化了梨果实石细胞中的木质素,并通过谱学方法对其特性进行了研究。上述两种来源的木质素结构类型基本一致,但提取的‘幸水’木质素重均分子量(WMW),分子量分散系数(MWD)都要大于‘砀山酥梨’。相应地,其玻璃化温度(GTT)也要高于‘砀山酥梨’,为160℃。石细胞木质素的紫外光谱吸收表现出其典型的苯环结构,并在红外光谱上得到验证。综合Maule试剂染色、红外光谱和硝基苯氧化产物HPLC分析的结果：发现石细胞木质素含有大量的愈创木基单元和一部分紫丁香基结构单元,而对羟基苯丙烷结构单元很少。进一步结合对1H-NMR和13C-NMR波谱的分析,初步明确梨果实石细胞木质素为典型的双子叶植物木质素,但其愈创木基结构单元含量高于其他树种,愈创木基与紫丁香基结构单元比例(G／S)高达4,接近于裸子植物,表现出软木木质素的结构特性。
三、梨果实石细胞在幼果中分布密度最高,此后因果实生长和膨大的“稀释”作用,其分布密度逐渐降低。石细胞团的大小随果实的生长一直增大。苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)和多酚氧化酶(PPO)的活性变化规律基本一致,其活性高峰都出现在幼果发育期,后随着果实发育逐渐下降。套袋处理能够抑制‘砀山酥梨’果实石细胞的发育,而且套袋越早效果越显著,相应PAL、POD、PPO的活性也较低。通过对比不同时期套袋处理的不同发育阶段果实内酶活性的平均值与其相应的石细胞最终含量,发现梨果实石细胞最终含量与POD、PAL、PPO活性之间呈正相关关系,相关系数分别为：RPOD=0.8035; RPAL=0.8134; RPPO=0.9186。进一步对‘砀山酥梨’进行喷CaCl2和多效唑处理,并以‘幸水’为品种对照,发现在两个品种上钙处理均能降低果实中木质素、石细胞含量和PAL、POD、PPO的总活性,而多效唑则起着相反的作用。酶活性和木质素、石细胞含量之间成正相关关系,并且细胞壁结合过氧化物酶(PODⅡ)在石细胞发育过程中起更大的作用。
四、以‘砀山酥梨’为试验材料,研究了梨果实中过氧化物酶(POD)、过氧化氢(H2O2)与石细胞及木质素之间的关系。成熟‘砀山酥梨’果肉干重的36.03%为石细胞,15.23%为木质素,其中有70.6%的木质素存在于石细胞中,石细胞重量的29.83%为木质素。通过徒手切片对果实中石细胞及过氧化物酶进行组织染色定位,发现无论是在横切面还是纵切面上,两者的分布区域都保持一致。木质素在果实发育初期大量合成,而细胞质过氧化物酶(PODⅠ)和细胞壁结合过氧化物酶(PODⅡ)都随果实的发育而逐渐降低,但PODⅡ对木质素单体类似物丁香醛连氮(Syringaldazine)的催化能力要远远高于POD I. POD和H2O2在梨果实细胞中的分布主要集中在细胞壁和细胞间隙之中。进一步通过DEAE离子交换层析柱分离PODⅡ中的碱性和酸性PODⅡ,并进行底物特异性分析,发现PODⅡ对Syringaldazine的催化主要由碱性PODⅡ完成。在梨果实中极有可能是碱性的细胞壁结合过氧化物酶(PODⅡ)在过氧化氢的存在下参与了木质素合成的最后一步反应,促进了石细胞的形成。
五、提取了‘砀山酥梨’及‘幸水’果肉总RNA,并通过RT-PCR、克隆和测序,获得了苯丙氨酸解氨酶(PAL),过氧化物酶(POD)和肉桂醇脱氢酶(CAD)的cDNA片段。在核苷酸水平上,2个品种PAL基因片段大小为653bp,只有3个碱基的差异,编码1个完全相同的氨基酸序列,该序列由217个残基组成。所推导的氨基酸序列与西洋梨Pyrus communis (ABB70117)、甜樱桃Prunus avium (AAC78457)、树莓Rubus idaeus (AAF40224)和野生橘Citrus clementina×Citrus reticulata(CAB42793)氨基酸序列的相似性分别达到98.6%、98.6%97.7%和97.2%。此外,它们含有4个与其他PAL蛋白质相似的蛋白质保守活性位点。
两个品种CAD基因的cDNA片段大小为689bp,核苷酸序列同源性为99.4%,有4个碱基的差异,而氨基酸序列相似性为99.6%,只有1个氨基酸残基的差异。所推导的氨基酸序列与苹果Malus domestica (AAC06319)、梅Prunus mume (BAE48658)、枇杷Eriobotrya japonica (ABV44810)和葡萄Vitis vinifera(CA021890)氨基酸序列的相似性分别达到97.8%、95%、92.6%和83.4%。
两个品种POD基因的cDNA片段大小约为574bp,编码191个氨基酸残基,它们之间的核苷酸序列和氨基酸序列没有任何差异。推导的氨基酸序列与烟草Nicotiana tabacum (AAD33072)、文心兰Oncidium Gower Ramsey (ABC02343)、盐芥Thellungiella halophila (ABU54828)和拟南芥Arabidopsis thaliana (CAA6686)氨基酸序列的相似性分别为84.3%、86.4%、86.9%和86.4%。此外,它们含有与其他POD蛋白相似的3个保守活性位点和2个底物结合位点。
结果表明,本研究克隆到的PB-PAL(登录号：FJ478149). PP-PAL(登录号：FJ478150). PB-CAD(登录号：FJ478151). PP-CAD(登录号：FJ478152). PB-POD(登录号：FJ478153)和PP-POD(登录号：FJ478154)就是梨果实中的PAL、CAD和POD基因片段。
Sclereid or stone cell is one of the crucial factors impacting grit texture in pear fruit. It is reported that genetic background is the intrinsic factor for sclereids development; however the culture practices can also influence their development consumingly. Unfortunately, no great progress is achieved on pear sclereids research till now; researchers always omit lignin-the chemical nature of sclereids, and few papers are found focusing on pear sclereid lignin directly. In this study,'Dangshansuli'pear which contains high sclereid content was used as the major material, and'Kousui'which has low content of sclereid as accessorial material to characterize sclereid structure and composition; also, PAL (Phenylalanine Ammonia Lyase), CAD (Cinnamyl Alcohol Dehydrogenase) and POD (Peroxidase) genes were cloned and analysed. The results are as follows:
1. Anatomy, ultrastructure and lignin distribution in stone cells from the fruit of two Pyrus species (Pyrus bretschneideri cv. Dangshansuli and Pyrus pyrifolia cv. Kousui) were examined by using light microscopy (LM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray energy spectrometer and inductive coupled plasma emission spectrometer (ICP) as well as autofluorescence analysis. Sections stained with phloroglucinol-HCl revealed the presence of lignin in stone cells, and showed that stone cells were distributed in a mosaic pattern in the flesh, with larger stone cells concentrated around the core and smaller ones in the pericarp. There were no obvious differences in stone cell structure between the two varieties, but stone cell size and content was much greater in'Dangshansuli'than in'Kousui.'Further, lignin accounted for 29.8% of stone cell composition in'Dangshansuli', a significantly higher proportion than that in 'Kousui'(24.6%), basing on acetyl bromide method; this result was confirmed by autofluorescence analysis. More detailed information on lignin distribution across the cell wall was obtained by TEM combined with the KMnO4 staining technique. In TEM images, cell walls of both pear varieties were typically divided into four layers:compound middle lamella (CML), secondary wall 1 (S1), outer secondary wall 2 (S2L) and secondary wall 2 (S2), with different staining intensities for different lignin concentrations in those regions.
2. Lignin isolated from sclereids of'Dangshansuli'and'Kousui'pear were studied with several spectroscopies, such as ultra violet spectroscopy (UV), fourier transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC),1H nuclear magnetic resonance (1H-NMR) and 13C nuclear magnetic resonance (13C-NMR). The lignin were the same in structural information, however the weight-average molecular weight (WMW) and molecular weight distribution (MWD) of'Kousui'lignin both were higher than 'Dangshansuli'lignin, consequently, it had higher glass transition temperature (GTT). Typical benzene structure showed in UV spectra was also confirmed by IR spectra. Great vanillin and some syringaldehyde found in lignin nitrobenzene oxidation products and Maule reagent staining result indicated the presence of guaiacyl unit in the lignin. Further, with the aids of 1H and 13C-NMR spectra, lignin in pear sclereids was characterized as typical guaiacyl-syringyl lignin (G-S lingnin), however it has higher content guaiacyl unit than other angiosperm, the ratio for guaiacyl and syringyl unit was as high as 4, more closely to gymnosperm.
3. Distribution density of sclereids was highest in pear fruitlet and then descended with fruit development; however the size of sclereid cluster increased gradually till fruit mature. With regards to phenylalanine ammonia lyase (PAL), peroxidase (POD) and polyphenol oxidase (PPO), their activity peaks always appeard in fruitlets, and then declined gradually in the course of fruit development. Fruit bagging had good effects on'Dangshansuli'fruit quality; it can reduce the content and size of sclereid in mature fruit; furthermore, bagging stage had influence on the effects, the earlier the better. Also, CaCl2 showed the same effects as fruit bagging on both'Dangshansuli'and'Kousui', while PP333 had the opposite result. All the data from this section showed a positive relationship between the enzymes' activities and lignin, sclereid content and size. Especially the cell wall bounded peroxidase (PODⅡ) played more important role in cell lignification.
4. The relationship among peroxidase, hydrogen peroxide, lignin and sclereid was studied by using'Dangshansuli'as material. Sclereids accounted for a big amount in dry fruit weight as 36.03%, and they contained 29.83%lignin which accounted for 70.6%of the total in fruit. Peroxidase activity was found almost around sclereids in free hand sections, and in the cell wall at cellular level. Besides, hydrogen peroxide was also localized at the similar regions, such as cell wall, cell corner and intercellular layer. Lignin was synthesized in big scale along with a burst of hydrogen peroxide in young fruit; also the soluble peroxidase (PODⅠ) and cell wall bounded peroxidase (PODⅡ) showed the same changing tendency, however the PODⅡalways possessed higher affinity to syringaldazine-an analog of lignin monomer. Further study basing on substrate affinity analysis demonstrated that it was the basic PODⅡshowing high catalytic efficiency. Lignin biosynthesis and sclereid development were related to basic PODⅡ.
5. Two PAL genes, two POD genes, and two CAD genes were cloned and sequenced using RT-PCR from'Dangshansuli'and'Kousui'pear respectively. In nucleotide sequence, the two PAL genes had 3 bp differences, and coded an identical amino acid sequence which comprised of 217 residues. Meanwhile, the amino acid sequence of the two PAL was 98.6%identical to PAL from Pyrus communis (ABB70117),98.6% identical to PAL from Prunus avium (AAC78457),97.7%identical to PAL from Rubus idaeus (AAF40224), and 97.2%indentical to PAL from Citrus clementina×Citrus reticulata (CAB42793) respectivity. Further, they were found to have 4 similar active sites with PAL from other plant species.
The two CAD genes had 4 bp differences resuting in one residue differences. The deduced amino acid sequence of the two CAD was 97.8%identical to CAD from Malus domestica (AAC06319),95%identical to CAD from Prunus mume (BAE48658), 92.6%identical to CAD from Eriobotrya japonica (ABV44810) and 83.4%identical to CAD from Vitis vinifera (CAO21890) respectively.
The two POD genes were identical in both nucleotide sequences and deduced amino acid sequences. Deduced amino acid sequence of the POD from pear was 84.3% identical to POD from Nicotiana tabacum (AAD33072),86.4%identical to POD from Oncidium Gower Ramsey (ABC02343),86.9%identical to POD from Thellungiella halophila (ABU54828) and 86.4%identical to POD from Arabidopsis thaliana (CAA6686) respectively. Moreover, POD from these two pears was found to have 3 similar active sites and 2 substrate bounding sites with POD from other plant species.
All the results from above showed PB-PAL (Accession No. FJ478149), PP-PAL (Accession No. FJ478150), PB-CAD (Accession No. FJ478151), PP-CAD (Accession No. FJ478152), PB-POD (Accession No. FJ478153) and PP-POD (Accession No. FJ478154) were the PAL, CAD and POD genes in pear fruit.
引文
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