一种高等植物小片段RNA分子标记方法

doi:10.3724/SP.J.1003.2012.08123

摘要/Abstract

摘要：

分子生物学技术特别是分子标记方法越来越多应用于研究植物间相似关系及其进化规律。为试图寻找一种适合缺乏分子生物学数据的, 以及能够在属以上高阶元间快速比较研究的小片段RNA分子标记方法, 作者通过含HAc-NaAc缓冲体系的SDS总核酸提取方法, 提取了53种高等植物的总核酸。将总核酸在7%的聚丙烯酰胺凝胶中电泳, 结果发现: (1)在100-200 bases的位置有高丰度的RNA条带, 条带稳定清晰, 结果可重复再现, 用DNase I和RNase A处理后认为这些片段属于小片段RNA。(2)比较了绿豆幼苗经0-8 h不同光照处理, 以及猕猴桃不同品种和器官之间的小片段RNA, 表明不同光照时间、不同性别的花蕾、叶片和染色体倍性间的带型一致, 说明这些片段部分具有一定的空间结构, 其表达极其稳定, 无明显差异。(3)同一科内的植物的带型存在多态性, 主要是由地理隔绝造成的差异, 并且分类阶元高的多态性百分比和多态性信息量高于分类阶元低的。(4)经过比较蕨类植物、裸子植物和被子植物53种植物的小片段RNA, 发现在100-200 bases中含3-6条清晰的条带, 带型跟植物亲缘关系相关。因此, 作者认为100-200 bases位置的小片段RNA可作为鉴定植物科间和科内亲缘关系的一种分子辅助标记。

关键词: SDS法, 总核酸, 小分子RNA, 亲缘关系, 分子标记

Abstract

In recent years, molecular biology techniques, especially molecular markers, have been applied to the study of species similarity and evolutionary relationships in the field of plant systematics. Here, we attempted to find a small fragment of RNA that could be used as a molecular marker for the rapid identification of plant taxa above genus. To do so, total nucleic acids were isolated from 53 species of higher plants using SDS extracting solution containing HAc-NaAc buffer. The isolated nucleic acids were subjected to 7% polyacrylamide gel electrophoresis (PAGE). Results showed: (1) A large number of bands, which were at the length of 100-200 bases and with clear shape. The bands patterns were stable and repeatable. These segments were confirmed to be short RNAs by DNaseI and RNaseA treatment; (2) No obvious differences in band patterns were found among different varieties, organs, sexualities and ploidy of kiwifruit seedlings. A similar case existed in mung bean seedlings after different light treatment periods; (3) Banding patterns of plants in the same family were polymorphic, with differences mainly related to geographic isolation. Higher taxonomic categories had a higher percentage of polymorphic band (PPB) and higher values of polymorphism information content (PIC) than lower taxonomic categories; (4) Three to six clear bands with a length of 100-200 bases, whose band patterns were related to the phylogenic relationship, were found through comparison of short RNAs from 53 species of ferns, gymnosperms and angiosperms. Thus, short RNAs with a length of 100-200 bases showed potential as a molecular marker for determining inter- or intrafamily phylogenic relationships.

Key words: SDS method, total nucleic acid, short RNA, phylogenic relationship, molecular markers

刘文, 梁红, 杨碧忍, 林杰文, 胡延吉 (2012) 一种高等植物小片段RNA分子标记方法. 生物多样性, 20, 86-93. DOI: 10.3724/SP.J.1003.2012.08123.

Wen Liu, Hong Liang, Biren Yang, Jiewen Lin, Yanji Hu (2012) Short RNAs, potential novel molecular markers for higher plants. Biodiversity Science, 20, 86-93. DOI: 10.3724/SP.J.1003.2012.08123.

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链接本文: https://www.biodiversity-science.net/CN/10.3724/SP.J.1003.2012.08123

https://www.biodiversity-science.net/CN/Y2012/V20/I1/86

图/表 7

表1 本实验所采用的植物材料

Table 1 Plants used in the present study

分类阶元 Taxonomic category		中文名 Chinese name	拉丁名 Scientific name
被子植物门 Angiospermae
双子叶植物纲 Dicotyledononeae	猕猴桃科 Actinidiaceae	中华猕猴桃品种红阳♂	A. chinensis ‘Hongyang’♂
		中华猕猴桃品种红阳♀	A. chinensis ‘Hongyang’♀
		中华猕猴桃品种武雄♂	A. chinensis ‘Wuxiong’♂
		中华猕猴桃品种武植3号♀	A. chinensis ‘Wuzhi No.3’♀
		美味猕猴桃品种帮增1号♂	A. chinensis var. deliciosa‘Bangzeng No.1’♂
		美味猕猴桃品种和平1号♀	A. chinensis var. deliciosa ‘Heping No.1’♀
		美味猕猴桃品种米良1号♀	A. chinensis var. deliciosa ‘Miliang No.1’ ♀
	桑科 Moraceae	大叶榕	Ficus altissima Blume
		水同木	Ficus fistulosa Reinw. ex Blume
		垂叶榕	Ficus benjamina L.
		柳叶榕	Ficus variolosa Lindl. ex Benth.
		细叶榕	Ficus microcarpa L. f.
		桂木	Artocarpus nitidus subsp.lingnanensis(Merr.) F. M. Jarrett
		面包树	Artocarpus communis J. R. Forst. et G. Forst.
		树菠萝	Artocarpus heterophyllus Lam.
	豆科 Leguminosae	红花羊蹄甲	Bauhinia blakeanaDunn
		酸豆	Tamarindus indica L.
		紫花苜蓿	Medicago sativa L.
		花生	Arachis hypogaeaL.
		柱花草	Stylosanthes scabra Vogel.
		黄槐	Cassia surattensis Burm. f.
		红绒球	Calliandra haematocephala Hassk.
		绿豆	Vigna radiata (L.) R. Wilczek
	夹竹桃科 Apocynaceae	夹竹桃	Nerium oleander L.
		鸡蛋花	Plumeria rubraL.
		狗牙花	Tabernaemontana divaricata (L.)R. Br. ex Roem. et Schult.
		长春花	Catharanthus roseus (L.) G. Don
		沙漠玫瑰	Adenium obesum (Forsk.) Roem. & Schult.
	木犀科 Oleaceae	茉莉	Jasminum sambac (L.) Aiton
	樟科 Lauraceae	樟树	Cinnamomum camphora (L.) J. Presl.
单子叶植物纲Monocotyledoneae	禾本科 Poaceae	玉米	Zea mays L.
		香根草	Chrysopogon zizanioides (L.) Roberty
		狗尾草	Setaria viridis (L.) P. Beauv.
		小麦	Triticum aestivum L.
		水稻	Oryza sativa L.
		佛肚竹	Bambusa ventricosa McClure
	棕榈科 Arecaceae	鱼尾葵	Caryota ochlandra Hance
	天南星科 Araceae	合果芋	Syngonium podophyllumSchott.
	鸭跖草科 Commeliaceae	鸭跖草	Commelina communisL.
裸子植物门 Gymnospermae	银杏科 Ginkgoaceae	银杏	Ginkgo bilobaL.
	苏铁科 Cycadaceae	华南苏铁	Cycas rumphiiMiq.
	罗汉松科 Podocarpaceae	罗汉松	Podocarpus macrophyllus (Thunb.) Sweet
	松科 Pinaceae	马尾松	Pinus massonianaD. Don
	南洋杉科 Araucariaceae	南洋杉	Araucaria cunninghamii Aiton ex D. Don
	杉科 Taxodiaceae	落羽杉	Taxodium distichum (L.) Rich.
	柏科 Cupressaceae	龙柏	Sabina chinensis(L.) Antoine ‘Kaizuca’
蕨类植物门 Pteridophyta	海金沙科 Lygodiaceae	海金沙	Lygodium japonicum (Thunb.) Sw.
	凤尾蕨科 Pteridaceae	井栏边草	Pteris multifidaPoir.
	铁角蕨科 Aspleniaceae	鸟巢蕨	Neottopteris nidus (L.) J. Sm.
	鹿角蕨科 Polypodiaceae	鹿角蕨	Platycerium wallichiiHook.
	肾蕨科 Davalliaceae	波士顿肾蕨	Nephrolepis exaltata (L.) Schott ‘Bostoniensis’

表1 本实验所采用的植物材料

Table 1 Plants used in the present study

分类阶元 Taxonomic category		中文名 Chinese name	拉丁名 Scientific name
被子植物门 Angiospermae
双子叶植物纲 Dicotyledononeae	猕猴桃科 Actinidiaceae	中华猕猴桃品种红阳♂	A. chinensis ‘Hongyang’♂
		中华猕猴桃品种红阳♀	A. chinensis ‘Hongyang’♀
		中华猕猴桃品种武雄♂	A. chinensis ‘Wuxiong’♂
		中华猕猴桃品种武植3号♀	A. chinensis ‘Wuzhi No.3’♀
		美味猕猴桃品种帮增1号♂	A. chinensis var. deliciosa‘Bangzeng No.1’♂
		美味猕猴桃品种和平1号♀	A. chinensis var. deliciosa ‘Heping No.1’♀
		美味猕猴桃品种米良1号♀	A. chinensis var. deliciosa ‘Miliang No.1’ ♀
	桑科 Moraceae	大叶榕	Ficus altissima Blume
		水同木	Ficus fistulosa Reinw. ex Blume
		垂叶榕	Ficus benjamina L.
		柳叶榕	Ficus variolosa Lindl. ex Benth.
		细叶榕	Ficus microcarpa L. f.
		桂木	Artocarpus nitidus subsp.lingnanensis(Merr.) F. M. Jarrett
		面包树	Artocarpus communis J. R. Forst. et G. Forst.
		树菠萝	Artocarpus heterophyllus Lam.
	豆科 Leguminosae	红花羊蹄甲	Bauhinia blakeanaDunn
		酸豆	Tamarindus indica L.
		紫花苜蓿	Medicago sativa L.
		花生	Arachis hypogaeaL.
		柱花草	Stylosanthes scabra Vogel.
		黄槐	Cassia surattensis Burm. f.
		红绒球	Calliandra haematocephala Hassk.
		绿豆	Vigna radiata (L.) R. Wilczek
	夹竹桃科 Apocynaceae	夹竹桃	Nerium oleander L.
		鸡蛋花	Plumeria rubraL.
		狗牙花	Tabernaemontana divaricata (L.)R. Br. ex Roem. et Schult.
		长春花	Catharanthus roseus (L.) G. Don
		沙漠玫瑰	Adenium obesum (Forsk.) Roem. & Schult.
	木犀科 Oleaceae	茉莉	Jasminum sambac (L.) Aiton
	樟科 Lauraceae	樟树	Cinnamomum camphora (L.) J. Presl.
单子叶植物纲Monocotyledoneae	禾本科 Poaceae	玉米	Zea mays L.
		香根草	Chrysopogon zizanioides (L.) Roberty
		狗尾草	Setaria viridis (L.) P. Beauv.
		小麦	Triticum aestivum L.
		水稻	Oryza sativa L.
		佛肚竹	Bambusa ventricosa McClure
	棕榈科 Arecaceae	鱼尾葵	Caryota ochlandra Hance
	天南星科 Araceae	合果芋	Syngonium podophyllumSchott.
	鸭跖草科 Commeliaceae	鸭跖草	Commelina communisL.
裸子植物门 Gymnospermae	银杏科 Ginkgoaceae	银杏	Ginkgo bilobaL.
	苏铁科 Cycadaceae	华南苏铁	Cycas rumphiiMiq.
	罗汉松科 Podocarpaceae	罗汉松	Podocarpus macrophyllus (Thunb.) Sweet
	松科 Pinaceae	马尾松	Pinus massonianaD. Don
	南洋杉科 Araucariaceae	南洋杉	Araucaria cunninghamii Aiton ex D. Don
	杉科 Taxodiaceae	落羽杉	Taxodium distichum (L.) Rich.
	柏科 Cupressaceae	龙柏	Sabina chinensis(L.) Antoine ‘Kaizuca’
蕨类植物门 Pteridophyta	海金沙科 Lygodiaceae	海金沙	Lygodium japonicum (Thunb.) Sw.
	凤尾蕨科 Pteridaceae	井栏边草	Pteris multifidaPoir.
	铁角蕨科 Aspleniaceae	鸟巢蕨	Neottopteris nidus (L.) J. Sm.
	鹿角蕨科 Polypodiaceae	鹿角蕨	Platycerium wallichiiHook.
	肾蕨科 Davalliaceae	波士顿肾蕨	Nephrolepis exaltata (L.) Schott ‘Bostoniensis’

表2 高等植物的小片段RNA多态性分析

Table 2 The short RNA segments polymorphism analysis in higher plants

分类阶元 Taxonomic category	材料数量 No. of species	同源片段数目 No. of band analogs	多态性位点数 No. of polymorphic loci	总位点数 No. of total loci	多态性百分比 PPB(%)	多态性信息量 PIC
禾本科 Poaceae	6	5-6	9	10	90	0.859
桑科 Moraceae	8	5	4	7	57.1	0.791
夹竹桃科 Apocynaceae	5	5	8	9	88.9	0.840
豆科 Leguminosae	7	5	17	17	100	0.926
蕨类植物门 Pteridophyta	5	3-4	12	12	100	0.889
裸子植物门 Gymnospermae	7	4-6	17	17	100	0.917

图1 部分植物总核酸和RNA电泳图谱。1: 鱼尾葵; 2: 细叶榕; 3: 垂叶榕; 4: 桂木; 5: 夹竹桃; 6: 猕猴桃雌株叶片总核酸; 7: 猕猴桃雄株叶片总核酸; 8: 猕猴桃雌花蕾总RNA; 9: 猕猴桃雄花总RNA; 10: 银杏总RNA; 11: 井栏边草总RNA。

Fig. 1 The electrophoretogram of the total nucleic acid and RNA from certain plants. 1, Caryota ochlandra; 2, Ficus microcarpa; 3, F. benjamina; 4, Artocarpus lingnanensis; 5, Nerium oleander; 6, Total nucleic acid from Actinidia chinensis(♀, leaf); 7, Total nucleic acid from Actinidia chinensis(♂, leaf); 8, Total RNA from Actinidia chinensis(♀, bud); 9, Total RNA from Actinidia chinensis(♂, bud); 10, Total RNA from Ginkgo biloba; 11, Total RNA from Pteris multifida.

图2 不同光照时间对绿豆叶片和小片段RNA的影响。A至F分别为每天光照0、1、2、4、6、8 h处理的叶片, 连续7 d, 2,000 lx, 25℃; 1-6分别为0、1、2、4、6、8 h光照处理7 d后提取的小片段RNA, M为单链RNA marker。

Fig. 2 The influence of illumination on the leaf of mung bean and short RNA segments. A to F show the leaves of mung bean after 0, 1, 2, 4, 6, 8 hours illumination each day, respectively (for seven days, 2,000 lx, 25℃); 1-6 show the short RNA segments after 0, 1, 2, 4, 6, 8 hours illumination each day for seven days. M is the single strand RNA marker.

图3 猕猴桃不同品种和器官的小片段RNA电泳图谱。1-7分别为红阳♂、武雄♂、帮增1号♂、红阳♀、武植3号♀、米良1号♀、和平1号♀(总RNA经过70℃ 10 min变性处理); 8-11分别是: 武植3号雌株花蕾、武植雄株花蕾、武植3号雌株叶片、武植雄株叶片(总RNA稀释1倍后, 不经过70℃变性处理)。M: 单链RNA marker。

Fig. 3 The electrophoresis result of short RNA segments from different kiwifruit varieties and organs. 1-7 areActinidia chinensis ‘Hongyang’ (♂), A. chinensis ‘Wuxiong’ (♂), A. chinensis var. deliciosa ‘Bangzeng No.1’ (♂), A. chinensis ‘Hongyang’ (♀), A. chinensis ‘Wuzhi No. 3’♀, A. chinensisvar. deliciosa ‘Miliang No.1’ (♀), A. chinensisvar. deliciosa ‘Heping No.1’ (♀), respectively. The total RNA samples which had been hold in 70℃for 10 minutes. 8-11 are A. chinensis ‘Wuzhi No. 3’ (♀) bud, A. chinensis ‘Wuxiong’ (♂) bud, A. chinensis ‘Wuxiong’ (♂) leaf, A. chinensis ‘Wuzhi No. 3’ (♀) leaf, respectively. The total RNA samples which had 1-fold dilution and had not been hold in 70 ℃. M is the single strand RNA marker.

图4 高等植物的小片段RNA图谱及其聚类分析图。A1: 玉米; A2: 狗尾草; A3: 香根草; A4: 小麦; A5: 水稻; A6: 佛肚竹。B1: 大叶榕; B2: 水同木; B3: 垂叶榕; B4: 柳叶榕; B5: 细叶榕; B6: 桂木; B7: 面包树; B8: 树菠萝。C1: 夹竹桃; C2: 鸡蛋花; C3: 狗牙花; C4: 长春花; C5: 沙漠玫瑰。D1: 红花羊蹄甲; D2: 酸豆; D3: 紫花苜蓿; D4: 花生; D5: 西卡柱花草; D6: 黄槐; D7: 红绒球。E1: 海金沙; E2: 鹿角蕨; E3: 鸟巢蕨; E4: 波士顿肾蕨; E5: 井栏边草。F1: 龙柏; F2: 马尾松; F3: 罗汉松; F4: 银杏; F5: 华南苏铁; F6: 落羽杉; F7: 南洋杉。M为单链RNA marker; 由上至下分别为200 bases和100 bases。

Fig. 4 The pattern and dendrogram of short RNA segments from higher plants. A1, Zea mays; A2, Setaria viridis; A3, Chrysopogon zizanioides; A4, Triticum aestivum; A5, Oryza sativa; A6, Bambusa ventricosa; B1, Ficus altissima; B2, Ficus harlandii; B3, Ficus benjamina; B4, Ficus variolosa; B5, Ficus microcarpa; B6, Artocarpus lingnanensis; B7, Artocarpus altilis; B8, Artocarpus heterophyllus, C1, Nerium oleander; C2, Plumeria rubra; C3, Ervatamia divaricata; C4, Catharanthus roseus; C5, Adenium obesum, D1, Bauhinia blakeana; D2, Tamarindus indica; D3, Medicago sativa; D4, Fabales, Arachis hypogaea; D5, Stylosanthes scabra; D6, Cassia surattensis; D7, Calliandra haematocephala; E1, Lygodium japonicum; E2, Platycerium wallichii; E3, Neottopteris nidus; E4, Nephrolepis exaltata‘Bostoniensis’; E5,Pteris multifida, F1, Sabina chinensis ‘Kaizuca’; F2, Pinus massomana; F3, Podocarpus macrophyllus; F4, Ginkgo biloba; F5, Cycas rumphii; F6, Taxodium distichum; F7, Araucaria cunninghamii. M is the single strand RNA marker; from up to down are 200 bases and 100 bases separately.

图5 11种被子植物的小片段RNA图谱。1-11分别为狗尾草、美人蕉、蒜、鱼尾葵、合果芋、鸭跖草、茉莉、中华猕猴桃、紫花苜蓿、狗牙花、樟树。M: 单链RNA marker。

Fig. 5 The pattern of short RNA segments from 11 species from Angiosperm. 1-11 areSetaria viridis,Canna indica, Allium sativum, Caryota ochlandra, Syngonium podophyllum, Commelina communis, Jasminum sambac, Actinidia chinensis, Medicago sativa, Ervatamia divaricata, and Cinnamomum camphora, respectively. M is the single strand RNA marker.

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